From owner-proteins@net.bio.net Mon Jan 01 22:00:00 1996 Path: biosci!bcm.tmc.edu!news.msfc.nasa.gov!newsfeed.internetmci.com!in1.uu.net!server-b.cs.interbusiness.it!usenet From: Giorgio Spagnol Newsgroups: bionet.molbio.proteins Subject: Redundancy in kinased prtotein detection Date: 2 Jan 1996 19:36:45 GMT Organization: University of Milan, dept. of Neurology Lines: 22 Message-ID: <4cc1gd$3mb@server-b.cs.interbusiness.it> NNTP-Posting-Host: bbs.galactica.it Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 1.1N (Macintosh; I; 68K) To: all X-URL: news:bionet.molbio.proteins Dear fellow researchers, I need to detect phosphotyrosinated proteins by western blotting, after receptor crosslinking of cells in culture. The only one protocol I have advice immunoprecipitation of cells lysate with mAb G410, followed by blotting, using the same mAB as primary antibody. Why not dyalise and concentrate the whole cell lysate, load it in the gel and immunoblot DIRECTLY, omitting the immunoprecipitation step? Any help would be greatly appreciated. Giorgio Spagnol, MD Institute of Neurology, Statal University, Milan, Via F. Sforza 35, 20122 Milan, Italy. Phone: 02-55190390 W, 02-6570326 H FAX: 02- 55190392 E-MAIL: spagnol@galactica.it From owner-proteins@net.bio.net Mon Jan 01 22:00:00 1996 Path: biosci!agate!sunsite.doc.ic.ac.uk!daresbury!not-for-mail From: dr y u sasidhar Newsgroups: bionet.molbio.proteins Subject: peptide aggregation Date: 3 Jan 1996 05:44:56 -0000 Lines: 15 Sender: lpddist@mserv1.dl.ac.uk Distribution: bionet Message-ID: <4cd54o$4vu@mserv1.dl.ac.uk> MIME-Version: 1.0 Original-To: proteins@dl.ac.uk dear netters, we are characterizing a beta hairpin peptide in aqueous solution using NMR. it appears that there is aggregation (conc 2mM). can solvents like methanol be used to prevent or decrease the extent of aggregation. are there any references for the same. any information on this will be greatly appreciated. Thanking you in advance, ramkrishna Ramkrishna dept. of chemistry iit bombay From owner-proteins@net.bio.net Mon Jan 01 22:00:00 1996 Path: biosci!ihnp4.ucsd.edu!swrinde!howland.reston.ans.net!news-e1a.megaweb.com!newstf01.news.aol.com!newsbf02.news.aol.com!not-for-mail From: rlh115@aol.com (RLH115) Newsgroups: bionet.molbio.proteins Subject: help with research project related to antibodies Date: 3 Jan 1996 01:20:22 -0500 Organization: America Online, Inc. (1-800-827-6364) Lines: 20 Sender: root@newsbf02.news.aol.com Message-ID: <4cd776$eao@newsbf02.news.aol.com> Reply-To: rlh115@aol.com (RLH115) NNTP-Posting-Host: newsbf02.mail.aol.com I am presently a student (with a biological research background) in a masters degree program in business administration. I am conducting a market survey to be used for a research paper to complete my degree requirements. The short one page questionaire examines the use of antibodies in all of the biological science fields. I would greatly appreciate the help of any individual who currently uses any antibodies for any reason to quickly take my questionaire. Since I am a student, I am unable to compensate anyone for their response. All individual information will be confidential. However, I am willing to provide any participant who requests it with a summary of the results upon completion of the project. If you are willing to assist me on my project, please e-mail me with a short request- " send survey" to: rlh115@aol.com Thank you for your time. R. Henderson The Pennsylvania State University From owner-proteins@net.bio.net Mon Jan 01 22:00:00 1996 Path: biosci!ihnp4.ucsd.edu!swrinde!howland.reston.ans.net!tank.news.pipex.net!pipex!oleane!jussieu.fr!univ-lyon1.fr!news.imag.fr!news.ujf-grenoble.fr!news-merci.ujf-grenoble.fr!macjesior.imag.fr!user From: jean-claude.jesior@imag.fr (J.C. JESIOR) Newsgroups: bionet.molbio.proteins Subject: New PPC version of 'FoldIt'‹ a protein modelling freeware Date: 2 Jan 1996 13:40:07 GMT Organization: Institut Albert BONNIOT, CNRS Lines: 116 Message-ID: NNTP-Posting-Host: macjesior.imag.fr Mime-Version: 1.0 Content-Type: text/plain; charset="iso-8859-1" Content-Transfer-Encoding: 8bit ANNOUNCING THE NEW PPC VERSION OF 'FOLDIT (LIGHT)' v.4.1.1 a molecular modelling freeware for Macintosh computers. €€€€€€€€€€€€€€€€€€€€€ FREEWARE €€€€€€€€€€€€€€€€€€€€€ €€€€ First release of the PPC version €€€€ DOWNLOAD * Using a WWW browser 'FoldIt (light)' can be downloaded from the following address: ftp://ftp.imag.fr/pub/TIMC/FoldIt.html * Using 'Fetch', it can be downloaded directly from: ftp.imag.fr in the directory: pub/TIMC *********************************************************************** You'll find two versions of the application: * Download the 'FoldIt (light) FAT' version if you are using a Power Mac or a Macintosh equipped with a 68LC040 CPU. * Download the 'FoldIt (light) FPU' version if you are using a machine equipped with a 68030 CPU (+68881/82 coprocessor) or with a full 68040 CPU (not a 68LC040!) *********************************************************************** WHAT 'FOLDIT (LIGHT)' DOES? 'FoldIt (light)' is a molecular modelling program to visualize and manipulate proteins. 'FoldIt (light)' is an interactive program with a user friendly interface. The goal of this program was to build an integrated environment in which statistical analysis as well 3D observations could be realized on PDB files without having to transfer files or swap machines. Our major underlying research project is still to try to improve the folding prediction methods (hence the name 'FoldIt'). This is also the sole desactivated feature in this released version (hence the adjective '(light)'). 'FoldIt (light)' is intented to provide the possibility to analyze proteins up to 1600 residues in size, to visualize and manipulate them interactively. It can directly read any protein coordinate text file from the Brookhaven Protein Data Bank (PDB) (Bernstein et al., 1977) including hetero-atoms and water molecules. 'FoldIt (light)' has two main windows: a color image window to display the protein structure and a text window to record the result of all operations requested by the user. The protein structure can be manipulated easily in real time with the mouse, zoomed or observed in stereo. Structure movement can also occur stepwise for a more precise control. Animations can be created. Steric conflicts, disulfide bonds, hydrogen and ionic interactions can be located and displayed in the protein structure. These interactions are also reported in the text window. Atoms and residues can be tagged individually (or globally) and structural information can then be extracted. Portions of a structure can be read into memory or displayed. Two structures can be read at the same time in memory and can be overlapped automatically. The second structure can be manipulated independently of the first. Bonds can be rotated and atomic parameters can be changed. The sequential folding of a protein can be simulated. It is possible to create a protein de novo from the menu or by entering the sequence from the keyboard. Structures can then be manipulated locally or forced into helices. The application can process structures in the batch mode to extract a number of structural features: Ramachandran plots, SS-bond plots, H-bond plots. Statistics on atomic parameters are displayed as histograms. The content of image and text windows as well as histograms can be saved to disk. SOFTWARE ENVIRONMENT 'FoldIt (light)' can only work on Macintosh computers running under system 7.0 and higher. It has been developed with Symantec Think Pascal and includes 52000 lines of code. The application can run in the background which is convenient for lengthy statistical procedures. 'FoldIt (light)' does not support printing but can save the content of its text window (report of different operations and data analysis) and the content of the image window (result of graphic operations) in TEXT or PICT formats. These files can then be manipulated by other specialized applications and printed. HARDWARE ENVIRONMENT 'FoldIt (light) FAT' runs on any Macintosh computers. For those who are using Macintosh computers equipped with 68030 (+ coprocessor) or 68040 microprocessors (i.e. Macintosh SE30 to Quadras), a version requirring FPU is also included in the package ('FoldIt (light) FPU'). The application itself requires 879 KB - 1.9 MB of memory and 3.3 KB of additional memory for each read residue: this means that 660 KB of extra RAM are necessary to read a typical 200-residue protein. Color monitors are preferred to give a better depth perception but black & white monitors may also be used. DOCUMENTATION A manual is included as a stand alone document which can be printed. Several small size protein structures are included as example. Balloon help is supported. ****************************************************** FoldIt (light) freeware by Jean-Claude JΙSIOR/ CNRS, Grenoble, France e-mail: jean-claude.jesior@imag.fr Version 4.1.1 - Sunday, 24 December 1995 developed with Symantec Think Pascal. FAT version compiled with Metrowerks Code Warrior. ****************************************************** -- Jean-Claude JESIOR jean-claude.jesior@imag.fr From owner-proteins@net.bio.net Mon Jan 01 22:00:00 1996 Newsgroups: bionet.molbio.proteins Path: biosci!galaxy.ucr.edu!ihnp4.ucsd.edu!swrinde!newsfeed.internetmci.com!in2.uu.net!sangam!iitb!powai!n1403305 From: n1403305@powai.cc.iitb.ernet.in (ramakrishna) Subject: peptide aggregation Message-ID: Date: Tue, 2 Jan 1996 16:17:33 GMT Organization: Computer Centre, Indian Institute of Technology, Bombay X-Newsreader: TIN [version 1.2 PL2] Lines: 11 dear netters, i am working on a beta hairpin peptide using NMR in aqueous solution. i have a feeling that the peptide is aggregating in aqueous solution. is there a way to prevent aggregation by adding some non polar solvents like methanol. does this help in the solubility and decreases the aggregation. any information on this will be greatly appreciated. thanks in advance, Ramkrishna From owner-proteins@net.bio.net Mon Jan 01 22:00:00 1996 Path: biosci!agate!ihnp4.ucsd.edu!swrinde!tank.news.pipex.net!pipex!sunsite.doc.ic.ac.uk!warwick!bham!usenet From: r.jefferis@bham.ac.uk (Roy Jefferis) Newsgroups: bionet.molbio.proteins Subject: Re: Protein Models & CAD/CAM Date: 2 Jan 1996 16:39:13 GMT Organization: University of Birmingham Lines: 21 Message-ID: <4cbn3h$74o@sun4.bham.ac.uk> References: <4a2rre$alp@agate.berkeley.edu> NNTP-Posting-Host: med340.bham.ac.uk X-Newsreader: WinVN 0.92.1 In article <4a2rre$alp@agate.berkeley.edu>, lhom@nature.Berkeley.EDU (Louis Hom) says: > >It seems to me that life for some folks would be easier if you could have >your computer create a real 3D representation of a protein structure. I >know that computer-controlled generation of prototypes in manufacturing is >done all the time. Does anyone know of someone trying to apply computer >aided manufacturing technology to protein structures? >-- >_______________________________________________________________________________ >Lou Hom >K '93 "Pe--li--gro . . . Pe-li-gro. . . >lhom@nature.berkeley.edu Peligro. Ah, peligro!" >http://www.ocf.berkeley.edu/~lhom **SPLAT!** Hello Lou, I have just begun enquiring into possiblities of generating protein models in this way. I am not a computer buff and at the moment I am trying to liase between buffs. We use quanta, on the Silicon Graphics, and I need to know whether we can generate a STL file (Stereolithography") for use on a CAD 5 system !!!!! Any comment ? From owner-proteins@net.bio.net Mon Jan 01 22:00:00 1996 Path: biosci!ihnp4.ucsd.edu!swrinde!newsfeed.internetmci.com!in1.uu.net!brighton.openmarket.com!decwrl!sunsite.doc.ic.ac.uk!peer-news.britain.eu.net!lyra.csx.cam.ac.uk!news From: Peter Wang Newsgroups: bionet.molbio.proteins Subject: Re: Metaloproteinase Date: 3 Jan 1996 01:43:20 GMT Organization: Centre for Protein Engineering Lines: 21 Message-ID: <4ccmvo$mvu@lyra.csx.cam.ac.uk> References: NNTP-Posting-Host: macx45.mrc-lmb.cam.ac.uk Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: quoted-printable X-Mailer: Mozilla 1.1N (Macintosh; I; 68K) To: gheavner@voicenet.com X-URL: news:gheavner.39.1A0FF4BF@voicenet.com gheavner@voicenet.com (George A. Heavner) wrote: >I would like your help in identifying a commercially available metaloproteinase inhibitor. >I have a series of experiments that I want to run in which I need to prevent a >cell surface protein from being cleaved from the surface of a cell by a >metaloproteinase. Specifically, I want to prevent membrane bound TNF from >being cleaved and appearing in the supernatant. Does anyone have experience >with a small molecule inhibitor that I could use? 1,10 phenanthroline should inhibit the cleavage of TNF from the cell surface; it inhibits cleavage (shedding) of TNF receptor, L-sel= ectin, and other surface proteins. It works by chelating metal ions needed by the protease. --------------------------------------------------------- Peter Wang, M.D., Ph.D. MRC Centre for Protein Engineering, Hills Road, Cambridge, CB2 2QH, England Tel (01223) 402104 (international calls +44-1223-402104) Fax (01223) 402140 ( " " +44-1223-402140) --------------------------------------------------------- From owner-proteins@net.bio.net Mon Jan 01 22:00:00 1996 Path: biosci!ihnp4.ucsd.edu!swrinde!newsfeed.internetmci.com!in2.uu.net!news.voicenet.com!philly05.voicenet.com!gheavner From: gheavner@voicenet.com (George A. Heavner) Newsgroups: bionet.molbio.proteins Subject: Metaloproteinase Date: Tue, 2 Jan 1996 19:40:21 LOCAL Organization: Voicenet - Internet Access - (215)674-9290 Lines: 12 Message-ID: NNTP-Posting-Host: philly05.voicenet.com X-Newsreader: Trumpet for Windows [Version 1.0 Rev B final beta #4] I would like your help in identifying a commercially available metaloproteinase inhibitor. I have a series of experiments that I want to run in which I need to prevent a cell surface protein from being cleaved from the surface of a cell by a metaloproteinase. Specifically, I want to prevent membrane bound TNF from being cleaved and appearing in the supernatant. Does anyone have experience with a small molecule inhibitor that I could use? Thanks George A. Heavner Senior Director, Peptide R&D Centocor, Inc. From owner-proteins@net.bio.net Tue Jan 02 22:00:00 1996 Path: biosci!CSHL.ORG!leemor From: leemor@CSHL.ORG (Leemor Joshua-Tor) Newsgroups: bionet.molbio.proteins Subject: Re: peptide aggregation Date: 3 Jan 1996 05:48:49 -0800 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 29 Sender: daemon@net.bio.net Distribution: world Message-ID: NNTP-Posting-Host: net.bio.net >dear netters, >we are characterizing a beta hairpin peptide in aqueous solution using >NMR. it appears that there is aggregation (conc 2mM). can solvents like >methanol be used to prevent or decrease the extent of aggregation. are >there any references for the same. any information on this will be greatly >appreciated. > >Thanking you in advance, >ramkrishna > >Ramkrishna >dept. of chemistry >iit bombay > > Those are notorius for aggregation, indeed. You may want to try TFE (trifluoroethanol) or you can try playing around with the pH, that sometimes has drastic affects. ******************************************************************* Leemor Joshua-Tor Cold Spring Harbor Laboratory Tel. (516) 367 8821 1 Bungtown Road Fax (516) 367 8873 Cold Spring Harbor, NY 11724 e-mail: leemor@cshl.org ******************************************************************* From owner-proteins@net.bio.net Tue Jan 02 22:00:00 1996 Path: biosci!POST.TAU.AC.IL!borovok From: borovok@POST.TAU.AC.IL (borovok ilya) Newsgroups: bionet.molbio.proteins Subject: I need any recommendations ... Date: 3 Jan 1996 06:33:54 -0800 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 40 Sender: daemon@net.bio.net Distribution: world Message-ID: References: <4cdicu$ha7@winx03.informatik.uni-wuerzburg.de> NNTP-Posting-Host: net.bio.net Dear Netters, I would like to ask you help me in seasrching any nice (possibly modern) review and other sources concerning a phenomenon of an iron binding by proteins. It's not very easy question. My case: I am a genetist and at present deal with a genetical analysis of Isopenicillin N Synthase (IPNS) from Streptomyces. It is non heme enzyme and requires Fe(II) as a cofactor. We did make mutations which switch off an activity of this enzyme and found all potential ligands which involved in iron-binding to form an active site; the same ligands have been shown recently in Nature (22 June, 95) for another IPNS from Aspergillus nidulans by use of 3D structure. So far this 3D structure is not placed in PDB. We and they did show there are two histidines and one aspartic acid those are ligands of this kind of dioxygenases. But in our case the 'total', triple, mutant IPNS protein with all switched off ligands did ... binds iron also very well. This unspecific (?) iron binding is same for both wild type protein and any mutant protein. This binding is very strong and an iron-IPNS-protein complex is running through Sephadex G-25 Fine keeping the same final ratio between iron and any mutant or wild type IPNS proteins. My additional questions are: a. Which reason(s) could explain(s) nicely our observation of an 'unspecific' iron binding by Isopenicillin N Synthase protein? b. Which kind of possible 'preventive' methods could be used to except such 'unspecific' iron binding by Isopenicillin N Synthase protein? Please, answering me, remember that my speciality is genetics! Please, be without very hard comments concerning my 'report'! Thank anybody in advance for any information. Ilya Borovok borovok@ccsg.tau.ac.il @post.tau.ac.il From owner-proteins@net.bio.net Tue Jan 02 22:00:00 1996 Path: biosci!biosci!not-for-mail From: gm@mole.bio.cam.ac.uk (Gillian Murphy \(Strangeways\)) Newsgroups: uk.jobs.offered,bionet.molbio.proteins Subject: Research Assistant - Strangeways Research Laboratory Date: 3 Jan 1996 19:36:43 -0800 Organization: University of Cambridge, England Lines: 20 Sender: daemon@net.bio.net Distribution: world Message-ID: <4cern0$mi0@lyra.csx.cam.ac.uk> NNTP-Posting-Host: net.bio.net Summary: Research Assistant position in Cambridge UK Keywords: Biochemistry,Molecular Biology,Metalloproteinase S T R A N G E W A Y S R E S E A R C H L A B O R A T O R Y RESEARCH ASSISTANT A post is available immediately to work on the binding mechanism of the tissue inhibitors of metalloproteinases. These inhibitors play key roles in both developmental processes and the course of degradative diseases. Studies involving chemical modification, protein engineering techniques and kinetic analyses are proposed. The position which is available for two years is suitable for an experienced graduate or a post-doctoral scientist from an appropriate field. Candidates with expertise in biochemistry or molecular biology are invited to apply. Further enquiries to Gillian Murphy on (01223) 243231 email gm@mole.bio.cam.ac.uk. Applications, including a c.v. and the names of two referees, should be made by 23rd February 1996 to the Administrative Secretary, Strangeways Research Laboratory, Worts Causeway, Cambridge CB1 4RN. From owner-proteins@net.bio.net Tue Jan 02 22:00:00 1996 Path: biosci!agate!howland.reston.ans.net!tank.news.pipex.net!pipex!peer-news.britain.eu.net!EU.net!Germany.EU.net!news.dfn.de!fu-berlin.de!news.belwue.de!News.Uni-Marburg.DE!news.th-darmstadt.de!uni-erlangen.de!winx03!wpxx02.toxi.uni-wuerzburg.de!not-for-mail From: krasel@wpxx02.toxi.uni-wuerzburg.de (Cornelius Krasel) Newsgroups: bionet.molbio.proteins Subject: Re: Metaloproteinase Date: 3 Jan 1996 09:31:10 GMT Organization: Dept. of Pharmacology, U Wuerzburg Lines: 12 Message-ID: <4cdicu$ha7@winx03.informatik.uni-wuerzburg.de> References: NNTP-Posting-Host: wpxx02.toxi.uni-wuerzburg.de X-Newsreader: TIN [UNIX 1.3 950824BETA PL0] George A. Heavner (gheavner@voicenet.com) wrote: > I would like your help in identifying a commercially available > metalloproteinase inhibitor. A mixture of 5 mM EDTA and 5 mM EGTA is often used to inhibit metalloproteases. --Cornelius. -- /* Cornelius Krasel, U Wuerzburg, Dept. of Pharmacology, Versbacher Str. 9 */ /* D-97078 Wuerzburg, Germany email: phak004@rzbox.uni-wuerzburg.de SP3 */ /* "Science is the game we play with God to find out what His rules are." */ From owner-proteins@net.bio.net Tue Jan 02 22:00:00 1996 Path: biosci!bcm.tmc.edu!news.msfc.nasa.gov!elroy.jpl.nasa.gov!swrinde!tank.news.pipex.net!pipex!newsfeed.internetmci.com!uwm.edu!msunews!netnews.upenn.edu!dsinc!eerie.acsu.buffalo.edu!ub!acsu.buffalo.edu!bdavidso From: bdavidso@acsu.buffalo.edu (Bruce A Davidson) Newsgroups: bionet.molbio.proteins Subject: Tubing material that doesn't bind proteins? Date: 3 Jan 1996 22:21:43 GMT Organization: UB Lines: 12 Message-ID: <4cevhn$st1@azure.acsu.buffalo.edu> NNTP-Posting-Host: orichalc.acsu.buffalo.edu NNTP-Posting-User: bdavidso I am looking for a tubing material that proteins do not bind to (at least exhibit very low binding). I am specifically interested in the material not binding to myoglobin. Thanks for your help. bdavidso@ubmedb.buffalo.edu Bruce Davidson Dept. of Anesthesiology SUNY@Buffalo From owner-proteins@net.bio.net Tue Jan 02 22:00:00 1996 Path: biosci!agate!usenet.kornet.nm.kr!news.kreonet.re.kr!news.dacom.co.kr!nntp.coast.net!chi-news.cic.net!news.uiowa.edu!usenet From: Nathan Baker Newsgroups: bionet.molbio.proteins,sci.chem,bionet.molec-model Subject: CHARMm Date: 3 Jan 1996 22:26:33 GMT Organization: University of Iowa, Iowa City, IA, USA Lines: 9 Distribution: world Message-ID: <4cevqp$ph2@flood.weeg.uiowa.edu> NNTP-Posting-Host: terminator.chem.uiowa.edu Xref: biosci bionet.molbio.proteins:6612 sci.chem:46235 bionet.molec-model:740 I've been having some trouble with getting CHARMm to read in coordinates from PDB files. I tried the mailing list, but it appears to be dead. Unfortunately, I do not have the graphical interface, Quanta. If anyone is familiar with CHARMm and willing to help me out, I would appreciate it. Thank you. Nathan Baker baker@terminator.chem.uiowa.edu From owner-proteins@net.bio.net Tue Jan 02 22:00:00 1996 Path: biosci!bcm.tmc.edu!news.msfc.nasa.gov!newsfeed.internetmci.com!usenet.eel.ufl.edu!usenet.cis.ufl.edu!usenet.ufl.edu!ppp-10-ts4.nerdc.ufl.edu!user From: dcvitkov@dental.ufl.edu (DGC) Newsgroups: bionet.molbio.proteins Subject: Searching Genbank for motifs Followup-To: bionet.molbio.proteins Date: 4 Jan 1996 04:58:12 GMT Organization: University of Florida Lines: 13 Distribution: world Message-ID: NNTP-Posting-Host: ppp-10-ts4.nerdc.ufl.edu Can anyone recommend a program that can search genbank or similar protein databases for short peptide motifs? I have tried BLAST but this program is definitely not appropriate. I would prefer to use something that has a Netscape or Mac interface. Thanks, Dennis Cvitkovitch, PhD Dept. of Oral Biology College of Dentistry U of Florida DCVITKOV@DENTAL.UFL.EDU From owner-proteins@net.bio.net Tue Jan 02 22:00:00 1996 Path: biosci!bcm.tmc.edu!news.msfc.nasa.gov!newsfeed.internetmci.com!howland.reston.ans.net!news-e1a.megaweb.com!newstf01.news.aol.com!newsbf02.news.aol.com!not-for-mail From: brucerat@aol.com (BruceRat) Newsgroups: bionet.molbio.proteins Subject: Linus Pauling's sickle cell electrophoresis expt. Date: 3 Jan 1996 23:34:02 -0500 Organization: America Online, Inc. (1-800-827-6364) Lines: 16 Sender: root@newsbf02.news.aol.com Message-ID: <4cflbq$d8v@newsbf02.news.aol.com> Reply-To: brucerat@aol.com (BruceRat) NNTP-Posting-Host: newsbf02.mail.aol.com Are you familiar with Linus Pauling's experiment demonstrating that the hemoglobin produced by heterozygotes for sickle cell anemia actually exists in two forms? He used gel electrophoresis, and demonstrated two peaks for heterozygotes vs one peak for homozygous recessives and another, single, peak for homozygous dominants. I am trying to do this experiment with my high school AP Biology students; especially since a teacher in our department is a carrier! I've no idea of the protocol, and have spent an 8-hour day proving that there are MANY ways to get absotutely no results. I'd appreciate guidance. Bruce Ratcliffe Edison-Computech High School Fresno, Ca (BruceRat@AOL.COM) From owner-proteins@net.bio.net Tue Jan 02 22:00:00 1996 Path: biosci!bloom-beacon.mit.edu!newsfeed.internetmci.com!chi-news.cic.net!nntp.coast.net!torn!ccshst05.cs.uoguelph.ca!ccshst01.cs.uoguelph.ca!jvrobert From: jvrobert@uoguelph.ca (Jennifer V Robertson) Newsgroups: bionet.molbio.proteins Subject: EGFr distribution Date: 4 Jan 1996 01:04:06 GMT Organization: University of Guelph Lines: 9 Message-ID: <4cf926$95o@ccshst05.cs.uoguelph.ca> NNTP-Posting-Host: ccshst01.cs.uoguelph.ca X-Newsreader: TIN [version 1.2 PL2] I'm looking for information that deals with EGFr distribution, particulary in the hair follicle. Any help would be greatly appreciated. Please reply to jvrobert@uoguelph.ca Thanks, Jen :) From owner-proteins@net.bio.net Tue Jan 02 22:00:00 1996 Path: biosci!agate!howland.reston.ans.net!swrinde!newsfeed.internetmci.com!uwm.edu!msunews!netnews.upenn.edu!cronkite.ocis.temple.edu!astro.ocis.temple.edu!driska From: driska@astro.ocis.temple.edu (Stephen P. Driska PhD) Newsgroups: bionet.molbio.proteins Subject: Re: NEPHGE and SDS transfer Date: 3 Jan 1996 19:34:25 GMT Organization: Temple University, Academic Computer Services Lines: 37 Message-ID: <4celo1$nt9@cronkite.ocis.temple.edu> References: <1995Dec30.162118.1@icbr.ifas.ufl.edu> NNTP-Posting-Host: astro.ocis.temple.edu X-Newsreader: TIN [version 1.2 PL2] rep@icbr.ifas.ufl.edu wrote: : Hey there, : Happy Holidays and all that. Posting this question for a friend. Has : anybody done NEPHGE gels and then transferred them under SDS conditions? I : seem to remember some posts about this quite awhile ago, although I think it : was just IEF and not NEPHGE. I would apreciate any replies concerning : incubations in transfer buffer or SDS containing buffer before blotting. : Basically, any help would probably be usefull. : Chuck Peterson : Whitney Lab. : St.Augustine, FL Do you mean A) transfering a 1-dimensional NEPHGE gel, or B) transferring a 2-D, NEPHGE/SDS gel? I haven't done A, but it should be like doing an IEF except that bands would be in different places. "B" is not really any different than a 2D IEF/SDS gels. The only thing that might be different is that with prolonged IEF, you might deplete the ampholytes in the gel, whereas in NEPHGE (usually run for a shorter time) you probably haven't lost as much ampho- lyte during the run. It just dawns on me now that you might mean you are worried about ampholytes binding to nitrocellulose/PVDF/nylon, etc. In that case, you might have problems. But let us know what the case is, and someone "out there" will probably be able to help..... good luck, Steve. -- Steve Driska, Physiology Department, Temple University Medical School Philadelphia, PA 19140 USA (215) 707-3283 driska@astro.ocis.temple.edu If I could think of something witty and clever to say, it would go right here ----->> ......................... From owner-proteins@net.bio.net Tue Jan 02 22:00:00 1996 Path: biosci!bcm.tmc.edu!news.msfc.nasa.gov!newsfeed.internetmci.com!in2.uu.net!newsfeed.ACO.net!news.univie.ac.at!news-admin@univie.ac.at From: "Bernd R. Binder" Newsgroups: bionet.molbio.proteins Subject: double-stranded linear DNA Date: 3 Jan 1996 21:45:07 GMT Organization: Clinical-Experimental Physiology Lines: 27 Message-ID: <4cetd3$g3g@ftp.univie.ac.at> NNTP-Posting-Host: cexp1.ephy.univie.ac.at Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 1.1N (Windows; I; 16bit) Hello netters! I have some questions, concerning double stranded DNA. Does somebody know where can I find or how to calculate: 1. The maximum bond angle or angle of rotation between neighbouring base pairs. Solvent - water and/or TAE 0,004 M, pH=8,0 2. The forces (energy) or funktion of: altering the angle of rotation from 5 to 0 degree 3. Energy to transform a random coiled DNA to a helical DNA with lowest potential energy (relation of radius of coil to angle of rotation for a given contour length). Segmental transformation is also possible. 4. The explicit value for the bending rigidity constant and likewise for the torsional rigidity constant. I would like to get these data formulated not in related quantities. It would be also helpful to get it with neglect of the anisotropy of the double helix. Thank You for giving hints to solve these problems. Dr. Manfred Peska, Institute of Medical Physiology University of Vienna E-mail: yuri.koshelnick@univie.ac.at From owner-proteins@net.bio.net Wed Jan 03 22:00:00 1996 Path: biosci!bcm.tmc.edu!news.msfc.nasa.gov!newsfeed.internetmci.com!tank.news.pipex.net!pipex!peer-news.britain.eu.net!warwick!bham!bhamcs!news.ox.ac.uk!news From: "Simon M. Brocklehurst" Newsgroups: bionet.molbio.proteins Subject: ANNOUNCE: NAOMI Upgrade Date: Thu, 04 Jan 1996 15:06:28 +0000 Organization: University of Oxford Lines: 76 Message-ID: <30EBECF4.1CFBAE39@bioch.ox.ac.uk> NNTP-Posting-Host: nmrv.ocms.ox.ac.uk Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 2.0b4 (X11; I; SunOS 4.1.3_U1 sun4m) NAOMI - Version Upgrade Announcement (Please note, NAOMI is provided at zero charge for academic use) (e-mail contact smb@bioch.ox.ac.uk) _____________________________________________________________________________ The computer program NAOMI Version 2.3b is available as of now from the NAOMI Web site at: http://www.ocms.ox.ac.uk/~smb/Software/N_details/naomi.html or via anonymous ftp ftp://nmrz.ocms.ox.ac.uk/pub/smb/naomi i.e. at nmrz.ocms.ox.ac.uk in directory pub/smb/naomi/ Users of versions older than 2.10 will need new license keys to allow the upgrade to work (please contact the author in this case). _____________________________________________________________________________ Upgrade features : Side-chain manipulations now include hydrogen atoms where appropriate. This makes for more seamless integration with X-PLOR (e.g. for molecular dynamics simulations, energy minimization, NMR structure calculation etc after manipulation of protein structures using NAOMI). _____________________________________________________________________________ What is NAOMI? NAOMI is an easy-to-use, state-of-the-art computer program which is aimed at both specialist and non-specialist researchers who make use of three-dimensional structures of proteins in their work. It has hundreds of users Worldwide. Some facilities offered by the program for working with structure include: automatic 'key' residue identification automatic hydrophobic core/packing analysis automatic hydrogen bonds main-chain and side-chain identification (including high quality energy calculations) automatic secondary structure (helix, strand and turn) classification using fuzzy logic automatic supersecondary structure classification (beta-hairpin loops) conformational parameters: phi,psi,chi1,chi2,chi3,chi4,chi5 etc solvent accessibility (both absolute and percentage) calculations automatic identification of disulphide bonds, salt bridges, chain-breaks side-chain modelling and manipulation applying symmetry operators automatic structure repair (building in missing atoms) NMR structure refinement module interfaces to graphics programs (MOLSCRIPT (and thus Raster3D), INSIGHT, QUANTA to allow automatic preparation of figures More details are available on the Web site. NB NAOMI currently works only on Silicon Graphics workstations running IRIX 5.* _____________________________________________________________________________ | | ,_ o Simon M. Brocklehurst, | / //\, Oxford Centre for Molecular Sciences, Department of Biochemistry, | \>> | University of Oxford, Oxford, UK. | \\, E-mail: smb@bioch.ox.ac.uk | WWW: http://www.ocms.ox.ac.uk/~smb/ |____________________________________________________________________________ From owner-proteins@net.bio.net Wed Jan 03 22:00:00 1996 Path: biosci!ihnp4.ucsd.edu!swrinde!howland.reston.ans.net!quagga.ru.ac.za!uct.ac.za!news From: Dennis Maeder Newsgroups: bionet.molbio.proteins Subject: Re: peptide aggregation Date: 4 Jan 1996 13:11:35 GMT Organization: Dept Biochemistry UCT Lines: 20 Message-ID: <4cgjm7$svn@groa.uct.ac.za> References: NNTP-Posting-Host: 137.158.134.7 Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 1.22 (Windows; I; 16bit) >i am working on a beta hairpin peptide using NMR in aqueous solution. >i have a feeling that the peptide is aggregating in aqueous solution. >is there a way to prevent aggregation by adding some non polar solvents >like methanol. does this help in the solubility and decreases the >aggregation. any information on this will be greatly appreciated. >thanks in advance, > >Ramkrishna > What is the sequence of your peptide? A beta haipin will leave only sidechains available for hydrogen bonding to water. If your peptide is apolar you may need to use a surfactant of sorts, but that may well make your NMR results less than useful. If however your peptide has some sidee-chain charge, you might want to exploit this by maximizing the expression of charge, but avoid neutrality under low-salt conditionswhere isoelectric precipitation will occur.i.e.work in >=50mM salt if possible. The N- and C- terminii may be charged as well, and these can be exploited. DennisMaeder From owner-proteins@net.bio.net Wed Jan 03 22:00:00 1996 Path: biosci!agate!howland.reston.ans.net!newsfeed.internetmci.com!in1.uu.net!fdn.fr!jussieu.fr!Newsmaster From: GURLIE Raphael Newsgroups: bionet.molbio.proteins,sci.chem,bionet.molec-model Subject: Re: CHARMm Date: Thu, 04 Jan 1996 13:04:06 +0100 Organization: Universites Paris VI/Paris VII - France Lines: 9 Message-ID: <30EBC236.1B32@glenan.urbb.jussieu.fr> References: <4cevqp$ph2@flood.weeg.uiowa.edu> NNTP-Posting-Host: glenan.urbb.jussieu.fr Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 2.0b4 (X11; I; HP-UX A.09.05 9000/712) To: Nathan Baker Xref: biosci bionet.molbio.proteins:6619 sci.chem:46288 bionet.molec-model:741 I am not familiar with CHARMm, but I've worked with JUMNA and X-PLOR and had some problems with PDB files. I had generated a PDB file with JUMNA and wanted to make a molecular dynamics simulation with X-PLOR, but X-PLOR coouldn't read my JUMNA PDB becaude nucleotide libraries are different between JUMNA and X-PLOR. If you can, get an example of a CHARMm PDB file and make sure yours can be red. If not you'll have to modify it.... Raphael From owner-proteins@net.bio.net Wed Jan 03 22:00:00 1996 Path: biosci!agate!newsxfer2.itd.umich.edu!newsfeed.internetmci.com!in2.uu.net!news00.sunet.se!sunic!news99.sunet.se!umdac!news From: Michael Daws Newsgroups: bionet.molbio.proteins Subject: Peptide immobilisation - Spacer length? Date: 4 Jan 1996 15:32:52 GMT Organization: UCMP Lines: 12 Message-ID: <4cgrv4$3sf@studium.student.umu.se> NNTP-Posting-Host: clsmac.ucmp.umu.se Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 1.1N (Macintosh; I; PPC) X-URL: news:bionet.molbio.proteins I am going to attach a peptide to an aminoalkyl agarose support using a bifunctional cross linker - does anyone know what the best spacer length would be to limit steric hindrance. Presumably if the hydrophobic spacer is too long it will allow increased non-specific binding or spacer cleavage? Thanks. Mike Daws From owner-proteins@net.bio.net Wed Jan 03 22:00:00 1996 Path: biosci!bcm.tmc.edu!news.msfc.nasa.gov!newsfeed.internetmci.com!newsserver.jvnc.net!synapse.bms.com!news-admin From: Stanley Krystek Newsgroups: bionet.molbio.proteins Subject: (no subject) Date: 4 Jan 1996 17:08:35 GMT Organization: Bristol-Myers Squibb Pharmaceutical Research Institute Lines: 14 Message-ID: <4ch1ij$c7j@synapse.bms.com> NNTP-Posting-Host: alcor.bms.com Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 1.1N (X11; I; IRIX 5.2 IP7) X-URL: news:bionet.molbio.proteins I am looking for software to map protease digest sites. Can someone suggest any software or programs available that provide a list of enzyme cleavage sites. thanks stan -- Stan Krystek Bristol-Myers Squibb krystek@alcor.bms.com From owner-proteins@net.bio.net Wed Jan 03 22:00:00 1996 Path: biosci!bcm.tmc.edu!news.msfc.nasa.gov!newsfeed.internetmci.com!in2.uu.net!news00.sunet.se!sunic!nntp.coast.net!col.hp.com!news.cis.okstate.edu!usenet From: price@vms.ocom.okstate.edu (Joseph A. Price, Ph.D.) Newsgroups: bionet.molbio.proteins Subject: Replacing acetonitrile Date: 4 Jan 1996 18:29:32 GMT Organization: COM-OSU Lines: 13 Message-ID: <4ch6ac$rsk@news.cis.okstate.edu> NNTP-Posting-Host: biochem1.ocom.okstate.edu Mime-Version: 1.0 X-Newsreader: WinVN 0.93.14 I have been told that someone published/found a mixture of alcohols that gave equivalent isolations as acn but are relatively nontoxic. Anyone know the mixture or paper?? -- Joseph A. Price, Ph.D price@vms.ocom.okstate.edu http://telemed1.ocom.okstate.edu/webpages/osteomed/depts/jprice.htm Ph. 918 561-8231 FAX 918 561 8412 Dept. Biochem. & Micro. COM-OSU 1111 W.17th. St. Tulsa OK 74107 From owner-proteins@net.bio.net Wed Jan 03 22:00:00 1996 Path: biosci!agate!howland.reston.ans.net!tank.news.pipex.net!pipex!peer-news.britain.eu.net!warwick!bham!usenet From: altabios@bham.ac.uk (John E. Fox) Newsgroups: bionet.molbio.proteins Subject: Re: Tubing material that doesn't bind proteins? Date: 4 Jan 1996 13:40:39 GMT Organization: Alta Bioscience Lines: 11 Message-ID: <4cglcn$t7e@sun4.bham.ac.uk> References: <4cevhn$st1@azure.acsu.buffalo.edu> NNTP-Posting-Host: bcs118.bham.ac.uk X-Newsreader: WinVN 0.90.6 In article <4cevhn$st1@azure.acsu.buffalo.edu>, bdavidso@acsu.buffalo.edu (Bruce A Davidson) says: > > >I am looking for a tubing material that proteins do not bind to (at least >exhibit very low binding). I am specifically interested in the material >not binding to myoglobin. My first choice would be PTFE, followed by PEEK. John Fox From owner-proteins@net.bio.net Wed Jan 03 22:00:00 1996 Path: biosci!bcm.tmc.edu!news.msfc.nasa.gov!newsfeed.internetmci.com!howland.reston.ans.net!paladin.american.edu!news.jhu.edu!NewsWatcher!user From: Neil.Clarke@qmail.bs.jhu.edu (Neil Clarke) Newsgroups: bionet.molbio.proteins,bionet.software Subject: WWW seq alignment site with "jumbling" test of significance? Date: 4 Jan 1996 19:49:08 GMT Organization: Johns Hopkins Lines: 12 Message-ID: NNTP-Posting-Host: 128.220.138.64 Xref: biosci bionet.molbio.proteins:6626 bionet.software:14347 Anyone know of a WWW site that does an alignment between two protein sequences with the option of rerunning the alignement using a randomized ("jumbled") sequence? as a test of significance? Having just given a lecture on sequence analysis during which I extolled the virtues of the Web, I was surprised to find that I couldn't find a good old-fashioned Needleman-Wunsch type program out there. Lots of local homology search algorithms and dynamic programming algorithms for database searching, but no 'optimal' sequence alignment stuff. Help, please! -- Neil Clarke neil.clarke@qmail.bs.jhu.edu From owner-proteins@net.bio.net Wed Jan 03 22:00:00 1996 Path: biosci!agate!news.ucdavis.edu!dcn88.dcn.davis.ca.us!user From: ijiwaru@wheel.dcn.davis.ca.us Newsgroups: bionet.molbio.methds-reagnts,bionet.molbio.proteins Subject: Re: fresh transformations for protein expression?? Date: Thu, 04 Jan 1996 23:40:14 +0500 Organization: University of California, Davis Lines: 37 Message-ID: References: <4ch87s$15oc@majestix.uni-muenster.de> NNTP-Posting-Host: dcn88.dcn.davis.ca.us X-Newsreader: Value-Added NewsWatcher 2.0b27.1+ Xref: biosci bionet.molbio.methds-reagnts:38230 bionet.molbio.proteins:6628 In article <4ch87s$15oc@majestix.uni-muenster.de>, Torsten Boerchers wrote: > Dear colleagues > > for quite some time we are successfully overexpressing > recombinant proteins in bacteria,mostly with the pET > system and mostly simple, nice, soluble proteins. > > The more we were surprised that we now on two occacions > with similar proteins encountered problems (i.e. no > expression) which were solved by freshly transforming > the E.coli (BL31(DE3)) with the expression vector instead > of starting from a plate. So fine, that works, but I would > nevertheless like to know whether others had similar > experiences and whether there is an explanation for this > behaviour available. I heared from some other folks routinely > doing fresh transformations but could not get a rationale. > > Looking forward to some ideas > Regards > > Torsten Tosten, I was told at some point in time that BL21 (and presumably the DE3 and DE3/pLysS strains also) don't hold plasmid very well and that I shouldn't even bother to keep frozen stocks of the transformed bacteria for expression purposes. I've always assumed that this may be due to leaky expression from the plasmid which may poison the bugs upon prolonged storage, although I have no evidence to support this claim. I'd like to know if you get a more lucid explaination. Lyle Najita Plant Pathology University of California - Davis From owner-proteins@net.bio.net Wed Jan 03 22:00:00 1996 Path: biosci!bcm.tmc.edu!news.msfc.nasa.gov!newsfeed.internetmci.com!uwm.edu!post.its.mcw.edu!usenet From: "Jennifer L. Potter" Newsgroups: bionet.molbio.proteins Subject: Driving Carboxypeptidases backwards Date: 4 Jan 1996 22:15:44 GMT Organization: Medical College of Wisconsin Lines: 26 Message-ID: <4chjig$6pe@post.its.mcw.edu> NNTP-Posting-Host: d350-1.biochem.mcw.edu Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 1.22 (Windows; I; 16bit) Dear Colleagues, I am thinking about adding a C-terminal tyrosine onto my protein of interest. To bypass all of the molecular biology, it was suggested that I simply drive a carboxypeptidase (probably either A or Y) reaction backwards to ADD a tyr instead of cleave the C-terminal residue (a Ser). The MERCK reference shows tyrosine to be rather insoluble (25 mM max in water) and mentions only that it is more soluble in alkaline solutions. I can't make a 1 M solution even dissolving it in Tris base (pH 10.5) and am therefore concerned about the feasibility of this idea because to drive a reaction backwards requires a great deal of the aa of interest (3 M according to a colleague). Any help re: solubility of tyrosine or ideas regarding the feasibility of this idea would be appreciated. Thanks in advance. Sincerely, Jennifer L. Potter jras@post.its.mcw.edu Medical College of WI Dept. of Biochemistry From owner-proteins@net.bio.net Wed Jan 03 22:00:00 1996 Path: biosci!bcm.tmc.edu!news.msfc.nasa.gov!newsfeed.internetmci.com!in1.uu.net!zib-berlin.de!fu-berlin.de!news.belwue.de!news.dfn.de!uni-muenster.de!news From: Torsten Boerchers Newsgroups: bionet.molbio.methds-reagnts,bionet.molbio.proteins Subject: fresh transformations for protein expression?? Date: 4 Jan 1996 19:02:20 GMT Organization: ICB Lines: 31 Message-ID: <4ch87s$15oc@majestix.uni-muenster.de> NNTP-Posting-Host: arnheim.uni-muenster.de Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 1.1N (Windows; I; 16bit) Xref: biosci bionet.molbio.methds-reagnts:38196 bionet.molbio.proteins:6625 Dear colleagues for quite some time we are successfully overexpressing recombinant proteins in bacteria,mostly with the pET system and mostly simple, nice, soluble proteins. The more we were surprised that we now on two occacions with similar proteins encountered problems (i.e. no expression) which were solved by freshly transforming the E.coli (BL31(DE3)) with the expression vector instead of starting from a plate. So fine, that works, but I would nevertheless like to know whether others had similar experiences and whether there is an explanation for this behaviour available. I heared from some other folks routinely doing fresh transformations but could not get a rationale. Looking forward to some ideas Regards Torsten ------------------------------------------------------------------------/ Torsten Boerchers / _/ _/_/_/ _/_/_/ Institute of Chemical- and Biochemical Sensor/ _/ _/ _/ _/ Research (Molecular Biology Group) / _/ _/ _/_/_/ Mendelstr 7, D-48149 Muenster, Germany / _/ _/ _/ _/ Fax: +49-251-980 2890 Phone: +49-251-980 2880/ _/ _/_/_/ _/_/_/_/ E-Mail: borcher@uni-muenster.de / -----------------------------------------------------------------/ From owner-proteins@net.bio.net Thu Jan 04 22:00:00 1996 Path: biosci!agate!howland.reston.ans.net!nntp.coast.net!news00.sunet.se!sunic!news99.sunet.se!news.uni-c.dk!eagle.novo.dk!usenet From: Lauge Schaffer Newsgroups: bionet.molbio.proteins Subject: Re: Peptide immobilisation - Spacer length? Date: Fri, 05 Jan 1996 16:50:01 -0800 Organization: Novo Nordisk A/S Lines: 16 Message-ID: <30EDC739.12B3@novo.dk> References: <4cgrv4$3sf@studium.student.umu.se> NNTP-Posting-Host: lsc.novo.dk Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 2.0b3 (Win16; I) Michael Daws wrote: > > I am going to attach a peptide to an aminoalkyl agarose support using a > bifunctional cross linker - does anyone know what the best spacer length > would be to limit steric hindrance. Presumably if the hydrophobic > spacer is too long it will allow increased non-specific binding or > spacer cleavage? > > Thanks. > > Mike Daws Depends on what your peptide is supposed to be doing after immobilization of course, but spacers of 6-12 atoms are commonly used. Lauge From owner-proteins@net.bio.net Thu Jan 04 22:00:00 1996 Path: biosci!bcm.tmc.edu!news.msfc.nasa.gov!newsfeed.internetmci.com!in1.uu.net!zib-berlin.de!fu-berlin.de!news.dfn.de!gs.dfn.de!uni-erlangen.de!winx03!wpxx02.toxi.uni-wuerzburg.de!not-for-mail From: krasel@wpxx02.toxi.uni-wuerzburg.de (Cornelius Krasel) Newsgroups: bionet.molbio.methds-reagnts,bionet.molbio.proteins Subject: Re: fresh transformations for protein expression?? Followup-To: bionet.molbio.methds-reagnts,bionet.molbio.proteins Date: 5 Jan 1996 09:06:32 GMT Organization: Dept. of Pharmacology, U Wuerzburg Lines: 25 Message-ID: <4cipmo$n0d@winx03.informatik.uni-wuerzburg.de> References: <4ch87s$15oc@majestix.uni-muenster.de> NNTP-Posting-Host: wpxx02.toxi.uni-wuerzburg.de X-Newsreader: TIN [UNIX 1.3 950824BETA PL0] Xref: biosci bionet.molbio.methds-reagnts:38233 bionet.molbio.proteins:6630 Torsten Boerchers (borcher@uni-muenster.de) wrote: > The more we were surprised that we now on two occacions > with similar proteins encountered problems (i.e. no > expression) which were solved by freshly transforming > the E.coli (BL31(DE3)) with the expression vector instead > of starting from a plate. In my and my colleagues' hands, the expression level in different clones of transfored BL21(DE3)pLysS can be quite different. Therefore we transform them, screen for clones which express the protein in high amounts and make a glycerol stock of this clone. It has happened once to a colleague of mine that he had to make a new glycerol stock because the old one didn't express sufficient amounts of protein any more. I have no idea why this might be the case. Maybe the levels of pLysS are different in different clones? --Cornelius. -- /* Cornelius Krasel, U Wuerzburg, Dept. of Pharmacology, Versbacher Str. 9 */ /* D-97078 Wuerzburg, Germany email: phak004@rzbox.uni-wuerzburg.de SP3 */ /* "Science is the game we play with God to find out what His rules are." */ From owner-proteins@net.bio.net Thu Jan 04 22:00:00 1996 Path: biosci!bcm.tmc.edu!news.msfc.nasa.gov!newsfeed.internetmci.com!news.sprintlink.net!usit.net!news From: showers@usit.net (Michael Showers) Newsgroups: bionet.molbio.proteins Subject: Help With Research Date: Sat, 06 Jan 1996 08:23:18 GMT Organization: United States Internet, Inc. Lines: 12 Message-ID: <4cl4l0$kfr@news.usit.net> NNTP-Posting-Host: jackson-slip12.dynamic.usit.net X-Newsreader: Forte Free Agent 1.0.82 My name is Todd Druen and I am currently an undergraduate student in microbiology. I am doing research on antibiotic resistant bacteria. I would like any information pertaining to the subject, literature, or tests to be run. Thank you for any help you can offer. Sincerely, Todd Druen E-Mail: showers@usit.net From owner-proteins@net.bio.net Thu Jan 04 22:00:00 1996 Path: biosci!bcm.tmc.edu!news.msfc.nasa.gov!newsfeed.internetmci.com!in2.uu.net!news00.sunet.se!sunic!news99.sunet.se!nntp-trd.UNINETT.no!daresbury!is.bbsrc.ac.uk!news From: Andy Phillips Newsgroups: bionet.molbio.methds-reagnts,bionet.molbio.proteins Subject: Re: fresh transformations for protein expression?? Date: 5 Jan 1996 14:10:10 GMT Organization: IACR-Long Ashton Lines: 31 Message-ID: <4cjbg2$93d@is.bbsrc.ac.uk> References: <4ch87s$15oc@majestix.uni-muenster.de> <4cipmo$n0d@winx03.informatik.uni-wuerzburg.de> NNTP-Posting-Host: pc0553.lars.bbsrc.ac.uk Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 1.22 (Windows; I; 16bit) Xref: biosci bionet.molbio.methds-reagnts:38241 bionet.molbio.proteins:6634 krasel@wpxx02.toxi.uni-wuerzburg.de (Cornelius Krasel) wrote: >In my and my colleagues' hands, the expression level in different >clones of transfored BL21(DE3)pLysS can be quite different. Therefore >we transform them, screen for clones which express the protein in >high amounts and make a glycerol stock of this clone. > >It has happened once to a colleague of mine that he had to make >a new glycerol stock because the old one didn't express sufficient >amounts of protein any more. > >I have no idea why this might be the case. Maybe the levels of >pLysS are different in different clones? This phenomenon isn't restricted to strains with pLysS. In a previous life, I expressed ribulose bisphosphate carboxylase (Rubisco) genes in JM83, and found that levels of enzyme activity varied enormously between individual isolates. Kept as a glycerol at -20oC, the level of expression we could get when using this to inoculate a fermenter declined over a period of months. We then regenerated a fresh, highly-expressing stock by re-transforming JM83. At the time, we speculated that the cause of this could be expression of the E.coli chaperonins, necessary for assembly of the Rubisco holoenzyme. Andy ----------------------------------------------------------------------------- Email : andy.phillips@bbsrc.ac.uk : University of Bristol Home : andy@cycad.demon.co.uk : IACR Long Ashton Research Station Phone : +44-275-392181 ext:257 : Long Ashton Fax : +44-275-394281 : Bristol, BS18 9AF, UK ----------------------------------------------------------------------------- From owner-proteins@net.bio.net Thu Jan 04 22:00:00 1996 Path: biosci!ns1.faseb.org!lamarck.sura.net!news.uky.edu!news From: Frank van de Loo Newsgroups: bionet.molbio.methds-reagnts,bionet.molbio.proteins Subject: Re: fresh transformations for protein expression?? Date: 5 Jan 1996 13:28:18 GMT Organization: University of Kentucky Lines: 22 Message-ID: <4cj91i$9mu@service2.uky.edu> References: <4ch87s$15oc@majestix.uni-muenster.de> NNTP-Posting-Host: 128.163.192.190 Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 1.22 (Windows; I; 16bit) To: borcher@uni-muenster.de Xref: biosci bionet.molbio.methds-reagnts:38240 bionet.molbio.proteins:6633 Torsten Boerchers wrote: > >The more we were surprised that we now on two occacions >with similar proteins encountered problems (i.e. no >expression) which were solved by freshly transforming >the E.coli (BL31(DE3)) with the expression vector instead >of starting from a plate. So fine, that works, but I would Me too: same problem, no explanation. However, I find I can avoid fresh transformations every time by making a a glycerol stock immediately, and then inoculating from this. I also went to the BLR strain (RecA-), but don't feel that this is any better. An aside: I spent quite a bit of effort learning how badly it can work to combine a T7lac (as opposed to plain T7) promoter and the pLysS-containing host. This potential problem is actually illustrated very nicely in Fig 2 on pg 25 of the Novagen cattledog. All the best, Frank van de Loo. From owner-proteins@net.bio.net Thu Jan 04 22:00:00 1996 Newsgroups: bionet.molbio.proteins,bionet.software Path: biosci!ihnp4.ucsd.edu!swrinde!newsfeed.internetmci.com!in2.uu.net!EU.net!peer-news.britain.eu.net!liv!news From: bates@liv.ac.uk (Andy Bates) Subject: DOS/Windows sequence software? Message-ID: X-Posted-From: InterNews 1.0.5@bates.bioc.liv.ac.uk Sender: bates@mailspool.liv.ac.uk Nntp-Posting-Host: bates.bioc.liv.ac.uk Organization: University of Liverpool X-Authenticated: bates on POP host mailspool.liv.ac.uk Date: Fri, 5 Jan 1996 10:19:49 GMT Lines: 27 Xref: biosci bionet.molbio.proteins:6632 bionet.software:14353 Sorry if this is a common request. I'm looking for some software to help teach students the delights of DNA (and protein) sequence analysis. It needs to be: 1. DOS/Windows 2. free 3. able to find restriction sites and search for short strings with mismatches/gaps 4. able to find ORFs and translate them 5. hopefully able to give some details of the hypothetical proteins, e.g. mol wt., amino acid composition. We currently use a simple menu-based DOS program call 'Mount & Conrad', which does all these things, but is not very colourful or flashy. I'm afraid this is a case where style may need to outweigh substance to some extent!! Any thoughts? Thanks a lot, Andy Bates. ____________________________________________________________ Dr. Andrew D. Bates Tel: (+44) (0)151 794 4322 Department of Biochemistry Fax: (+44) (0)151 794 4349 University of Liverpool Email: bates@liv.ac.uk PO Box 147 Liverpool L69 3BX, UK From owner-proteins@net.bio.net Thu Jan 04 22:00:00 1996 Path: biosci!bcm.tmc.edu!news.msfc.nasa.gov!newsfeed.internetmci.com!in1.uu.net!zib-berlin.de!fu-berlin.de!news.dfn.de!gs.dfn.de!uni-erlangen.de!winx03!wpxx02.toxi.uni-wuerzburg.de!not-for-mail From: krasel@wpxx02.toxi.uni-wuerzburg.de (Cornelius Krasel) Newsgroups: bionet.molbio.proteins,bionet.software Subject: Re: WWW seq alignment site with "jumbling" test of significance? Followup-To: bionet.molbio.proteins,bionet.software Date: 5 Jan 1996 09:08:31 GMT Organization: Dept. of Pharmacology, U Wuerzburg Lines: 14 Message-ID: <4cipqf$n0d@winx03.informatik.uni-wuerzburg.de> References: NNTP-Posting-Host: wpxx02.toxi.uni-wuerzburg.de X-Newsreader: TIN [UNIX 1.3 950824BETA PL0] Xref: biosci bionet.molbio.proteins:6631 bionet.software:14352 Neil Clarke (Neil.Clarke@qmail.bs.jhu.edu) wrote: > Anyone know of a WWW site that does an alignment between two protein > sequences with the option of rerunning the alignement using a randomized > ("jumbled") sequence? as a test of significance? I don't know of a web site, but I use Bill Pearson's FASTA package for this (compiled it myself on a Linux box). Works perfectly well. --Cornelius. -- /* Cornelius Krasel, U Wuerzburg, Dept. of Pharmacology, Versbacher Str. 9 */ /* D-97078 Wuerzburg, Germany email: phak004@rzbox.uni-wuerzburg.de SP3 */ /* "Science is the game we play with God to find out what His rules are." */ From owner-proteins@net.bio.net Thu Jan 04 22:00:00 1996 Path: biosci!bloom-beacon.mit.edu!newsfeed.internetmci.com!in1.uu.net!zib-berlin.de!fu-berlin.de!news.dfn.de!uni-muenster.de!news From: Torsten Boerchers Newsgroups: bionet.molbio.proteins,bionet.software Subject: Re: WWW seq alignment site with "jumbling" test of significance? Date: 5 Jan 1996 18:37:23 GMT Organization: ICB Lines: 21 Message-ID: <4cjr53$vak@majestix.uni-muenster.de> References: NNTP-Posting-Host: arnheim.uni-muenster.de Mime-Version: 1.0 Content-Type: text/plain; charset=iso-8859-1 Content-Transfer-Encoding: 8bit X-Mailer: Mozilla 1.1N (Windows; I; 16bit) To: Neil.Clarke@qmail.bs.jhu.edu Xref: biosci bionet.molbio.proteins:6637 bionet.software:14354 I haven΄t tried this place yet, since I always use the GCG package but I think this web-site should be what you΄re looking for http://genome.eerie.fr/fasta/align-query.html Good luck Torsten ------------------------------------------------------------------------/ Torsten Boerchers / _/ _/_/_/ _/_/_/ Institute of Chemical- and Biochemical Sensor/ _/ _/ _/ _/ Research (Molecular Biology Group) / _/ _/ _/_/_/ Mendelstr 7, D-48149 Muenster, Germany / _/ _/ _/ _/ Fax: +49-251-980 2890 Phone: +49-251-980 2880/ _/ _/_/_/ _/_/_/_/ E-Mail: borcher@uni-muenster.de / -----------------------------------------------------------------/ From owner-proteins@net.bio.net Thu Jan 04 22:00:00 1996 Path: biosci!agate!howland.reston.ans.net!nntp.coast.net!news00.sunet.se!sunic!news99.sunet.se!news.uni-c.dk!eagle.novo.dk!usenet From: Lauge Schaffer Newsgroups: bionet.molbio.proteins Subject: Re: Driving Carboxypeptidases backwards Date: Fri, 05 Jan 1996 16:56:19 -0800 Organization: Novo Nordisk A/S Lines: 15 Message-ID: <30EDC8B3.489F@novo.dk> References: <4chjig$6pe@post.its.mcw.edu> NNTP-Posting-Host: lsc.novo.dk Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 2.0b3 (Win16; I) Jennifer L. Potter wrote: > I am thinking about adding a C-terminal tyrosine onto my protein of > interest. To bypass all of the molecular biology, it was suggested > that I simply drive a carboxypeptidase (probably either A or Y) reaction > backwards to ADD a tyr instead of cleave the C-terminal residue (a Ser). Enzymatic coupling has been used extensively for trypsin. In this case you drive the reaction by running it in about 70% organic solvent (e.g. DMF/DMSO/1,4-Butanediol) which shifts the equilibrium towards synthesis. I don't know whether CpA or CpY works the same way (how about a little trip to the library?) Lauge From owner-proteins@net.bio.net Thu Jan 04 22:00:00 1996 Path: biosci!agate!spool.mu.edu!howland.reston.ans.net!EU.net!peer-news.britain.eu.net!lyra.csx.cam.ac.uk!news From: Manoj Ramjee Newsgroups: bionet.molbio.methds-reagnts,bionet.molbio.proteins Subject: Re: fresh transformations for protein expression?? Date: Fri, 05 Jan 1996 09:41:22 +0100 Organization: University of Cambridge Lines: 6 Message-ID: <30ECE432.4EB3@mole.bio.cam.ac.uk> References: <4ch87s$15oc@majestix.uni-muenster.de> NNTP-Posting-Host: molgen3.plantsci.cam.ac.uk Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 2.0b4 (Macintosh; I; 68K) Xref: biosci bionet.molbio.methds-reagnts:38232 bionet.molbio.proteins:6629 I used to work in a lab which encountered the same problem, but with yeast (S. cerevisiae). They put it down to recombination events that affected the expressed gene. I'm not sure about BL21 and its recombination ability, but could this be a possibility ? bYe. From owner-proteins@net.bio.net Thu Jan 04 22:00:00 1996 Path: biosci!bcm.tmc.edu!news.msfc.nasa.gov!newsfeed.internetmci.com!news.dacom.co.kr!news.uoregon.edu!news.orst.edu!ava.bcc.orst.edu!navarrer From: navarrer@ava.bcc.orst.edu (Roy Navarre) Newsgroups: bionet.molbio.proteins Subject: Protease inhibitors Date: 6 Jan 1996 01:17:24 GMT Organization: Oregon State University, Corvallis, Oregon Lines: 7 Message-ID: <4ckij4$bv7@news.orst.edu> NNTP-Posting-Host: ava.bcc.orst.edu Hi, does anyone know of any cell-permeable protease inhibitors? thanks Roy From owner-proteins@net.bio.net Thu Jan 04 22:00:00 1996 Newsgroups: bionet.molbio.proteins Path: biosci!rutgers!uwm.edu!vixen.cso.uiuc.edu!newsfeed.internetmci.com!howland.reston.ans.net!torn!utnut!oci!news From: Flip Hoedemaeker Subject: Re: Protein strange behaviour.HELP. Content-Type: text/plain; charset=us-ascii Message-ID: <30EDA969.41C6@oci.utoronto.ca> Sender: news@oci.utoronto.ca Content-Transfer-Encoding: 7bit Organization: Ontario Cancer Institute - U of Toronto References: <4cjvdb$hm8@server-b.cs.interbusiness.it> Mime-Version: 1.0 Date: Fri, 5 Jan 1996 22:42:49 GMT X-Mailer: Mozilla 2.0b2 (X11; I; IRIX 5.3 IP20) Lines: 23 Giorgio Spagnol wrote: > > Dear fellow researchers, > A glycoprotein we exctrated from brains reacts in immunoblot with a > monoclonal against its peptide moyety, as a 100 kD band. > Following 2D SDS-PAGE, however, the single band becomes two spots, same > MW but PI at 4.4 and 5.1, respectively. We think this might e due to > carbohydrate eterogeneicity and we will perform treatment syalidase > treatment, as soon as we get the enzyme. Apart from this we had had no > Ideas and we also have no reference on how to study such cases, so that > any suggestion would be greatly welcome. > Thanks in advance for your corteous attention. > Yes, it can be carbohydrate herterogeneity, but also a small sequence difference is possible. I worked on two isolectins, proteins of of about 35 kD. They were derived from the same gene but differential processing yielded two proteins with a one unit pI difference. The difference turned out to be only one Lys at the c-terminus. Amino acid sequencing is the only way to find these differences.... hope this helps. Flip From owner-proteins@net.bio.net Thu Jan 04 22:00:00 1996 Path: biosci!bcm.tmc.edu!news.msfc.nasa.gov!newsfeed.internetmci.com!netnews.nwnet.net!mule.fhcrc.org!news From: aelston Newsgroups: bionet.molbio.proteins Subject: Protein solution from tissue Date: 6 Jan 1996 00:31:30 GMT Organization: Fred Hutchinson Cancrer Research Center Lines: 12 Message-ID: <4ckft2$k8d@mule.fhcrc.org> NNTP-Posting-Host: 140.107.87.44 Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 1.22 (Windows; I; 16bit) Hi, My goal is to take tissue, put it into solution, and read it's collagen content using Sirius Red. My problem is the solution part! How can I get my tissue into solution without hydrolysis, without using concentrators, and keeping the final volume to <= 1 ml? Any suggestions? -ALE From owner-proteins@net.bio.net Thu Jan 04 22:00:00 1996 Path: biosci!agate!howland.reston.ans.net!swrinde!newsfeed.internetmci.com!in2.uu.net!server-b.cs.interbusiness.it!usenet From: Giorgio Spagnol Newsgroups: bionet.molbio.proteins Subject: Protein strange behaviour.HELP. Date: 5 Jan 1996 19:50:03 GMT Organization: University of Milan, dept. of Neurology Lines: 24 Message-ID: <4cjvdb$hm8@server-b.cs.interbusiness.it> NNTP-Posting-Host: bbs.galactica.it Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 1.1N (Macintosh; I; 68K) X-URL: news:bionet.molbio.proteins Dear fellow researchers, A glycoprotein we exctrated from brains reacts in immunoblot with a monoclonal against its peptide moyety, as a 100 kD band. Following 2D SDS-PAGE, however, the single band becomes two spots, same MW but PI at 4.4 and 5.1, respectively. We think this might e due to carbohydrate eterogeneicity and we will perform treatment syalidase treatment, as soon as we get the enzyme. Apart from this we had had no Ideas and we also have no reference on how to study such cases, so that any suggestion would be greatly welcome. Thanks in advance for your corteous attention. Giorgio Spagnol, MD Institute of Neurology, Statal University, Milan, Via F. Sforza 35, 20122 Milan, Italy. Phone: 02-55190390 W, 02-6570326 H FAX: 02- 55190392 E-MAIL: spagnol@galactica.it From owner-proteins@net.bio.net Fri Jan 05 22:00:00 1996 Path: biosci!ns1.faseb.org!lamarck.sura.net!newsfeed.internetmci.com!vixen.cso.uiuc.edu!usenet.ucs.indiana.edu!sunflower.bio.indiana.edu!gilbertd From: gilbertd@sunflower.bio.indiana.edu (Don Gilbert) Newsgroups: bionet.molbio.proteins,bionet.software Subject: Re: DOS/Windows sequence software? Date: 7 Jan 1996 02:09:16 GMT Organization: Biology, Indiana University - Bloomington Lines: 18 Message-ID: <4cna0c$nrc@usenet.ucs.indiana.edu> References: NNTP-Posting-Host: sunflower.bio.indiana.edu Xref: biosci bionet.molbio.proteins:6643 bionet.software:14359 seqpup covers all but #5 of your points, I believe. Find it at ftp://iubio.bio.indiana.edu/molbio/seqpup/ There should be a significant update out in the next couple of weeks. In article , Andy Bates wrote: >help teach students the delights of DNA (and protein) sequence >analysis. It needs to be: > >1. DOS/Windows >2. free >3. able to find restriction sites and search for short strings with >mismatches/gaps >4. able to find ORFs and translate them >5. hopefully able to give some details of the hypothetical proteins, >e.g. mol wt., amino acid composition. -- -- d.gilbert--biocomputing--indiana u--bloomington--gilbertd@bio.indiana.edu From owner-proteins@net.bio.net Sat Jan 06 22:00:00 1996 Path: biosci!SASK.USASK.CA!fang From: fang@SASK.USASK.CA Newsgroups: bionet.molbio.proteins Subject: Conference Date: 7 Jan 1996 15:37:44 -0800 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 9 Sender: daemon@net.bio.net Distribution: world Message-ID: NNTP-Posting-Host: net.bio.net Hi, netters. Is there any conference related to protein purification will be held in the States on February or March? Could you please inform me? Thank you in advance! -Li- From owner-proteins@net.bio.net Sat Jan 06 22:00:00 1996 Path: biosci!agate!uclink2.berkeley.edu!lhom From: lhom@uclink2.berkeley.edu (Louis George Hom) Newsgroups: bionet.molbio.proteins Subject: It's there, it's functional, but it's not detected Date: 7 Jan 1996 21:02:15 GMT Organization: University of California, Berkeley Lines: 1 Message-ID: <4cpccn$h09@agate.berkeley.edu> NNTP-Posting-Host: uclink2.berkeley.edu From owner-proteins@net.bio.net Sat Jan 06 22:00:00 1996 Path: biosci!bcm.tmc.edu!news.msfc.nasa.gov!newsfeed.internetmci.com!uwm.edu!msunews!harbinger.cc.monash.edu.au!lugb.latrobe.edu.au!newsmgr From: "R.K. Scopes" Newsgroups: bionet.molbio.methds-reagnts,bionet.molbio.proteins Subject: Re: fresh transformations for protein expression?? Date: 8 Jan 1996 06:08:43 GMT Organization: La Trobe University Lines: 23 Message-ID: <4cqcdb$39p@lugb.latrobe.edu.au> References: <4ch87s$15oc@majestix.uni-muenster.de> NNTP-Posting-Host: bcrs1.latrobe.edu.au Xref: biosci bionet.molbio.methds-reagnts:38338 bionet.molbio.proteins:6646 Torsten Boerchers wrote: > > Dear colleagues > > for quite some time we are successfully overexpressing > recombinant proteins in bacteria,mostly with the pET > system and mostly simple, nice, soluble proteins. > > The more we were surprised that we now on two occacions > with similar proteins encountered problems (i.e. no > expression) > We have had much the same experience, which apparently is very common, but not widely reported. It is unfortunate that unsuccessful experiments are not published: you could learn a lot ! (That's where the web is useful-this is one example). Expression deteriorates when colonies are kept on plates, and despite the plasmid still being there, it often can't be resusicated easily. We have tried several different hosts; some are better than others in this respect. Re-transform, or keep glycerol stocks of mid-log growing cells as a starter seems to be the best advice. Cheers. RKScopes From owner-proteins@net.bio.net Sun Jan 07 22:00:00 1996 Path: biosci!bcm.tmc.edu!news.msfc.nasa.gov!newsfeed.internetmci.com!tank.news.pipex.net!pipex!warwick!bham!bhamcs!news.ox.ac.uk!biomac7.jr2.ox.ac.uk!vurquidiI From: vurquidiI@molbiol.ox.ac.uk (Virginia Urquidi) Newsgroups: bionet.molbio.proteins Subject: Re: Searching Genbank for motifs Followup-To: bionet.molbio.proteins Date: Mon, 8 Jan 96 16:31:32 GMT Organization: Oxford University Lines: 21 Distribution: world Message-ID: References: NNTP-Posting-Host: biomac7.jr2.ox.ac.uk X-Newsreader: VersaTerm Link v1.1.6 In Article , dcvitkov@dental.ufl.edu (DGC) wrote: > >Can anyone recommend a program that can search genbank or similar protein >databases for short peptide motifs? I have tried BLAST but this program is >definitely not appropriate. I would prefer to use something that has a >Netscape or Mac interface. > I have used MOTIF (GCG Package) to search the PROSITE dictionary of protein sites and patterns and BLAST against the SBASE protein domain database for a 43 aa peptide. Check Nucleic Acids Res. 21, 1993 for more documentation. Hope this helps. V. Urquidi NDCB John Radcliffe Hospital Oxford - UK From owner-proteins@net.bio.net Sun Jan 07 22:00:00 1996 Path: biosci!NMU-GW.CC.NARAMED-U.AC.JP!mshima From: mshima@NMU-GW.CC.NARAMED-U.AC.JP (Midori Shima) Newsgroups: bionet.molbio.proteins Subject: (none) Date: 8 Jan 1996 01:26:41 -0800 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 2 Sender: daemon@net.bio.net Distribution: world Message-ID: <9601080916.AA04178@nmu-gw.cc.naramed-u.ac.jp> NNTP-Posting-Host: net.bio.net help From owner-proteins@net.bio.net Sun Jan 07 22:00:00 1996 Path: biosci!biosci!not-for-mail From: Davison@UH.EDU (Dan Davison) Newsgroups: bionet.molbio.proteins,bionet.software.gcg,bionet.software Subject: PIR Release 47 available for FTP, Gopher access Date: 8 Jan 1996 12:07:46 -0800 Organization: University of Houston Lines: 38 Sender: daemon@net.bio.net Distribution: bionet Message-ID: <4crcb5$mfo@masala.cc.uh.edu> NNTP-Posting-Host: net.bio.net Xref: biosci bionet.molbio.proteins:6650 bionet.software.gcg:1548 bionet.software:14373 PIR RELEASE 47 available The UH Gene-Server files have been updated with release 47 of the PIR. Anonymous ftp for the ascii and vms_lzw version is available on ftp.bchs.uh.edu in pub/gene-server/pir/pir_rel47/vms_lzw or pub/gene-server/pir/pir_47/ascii. Login as "anonymous" and send your e-mail address as the password. The conversion of files into the format needed for GCG is complete and the files are in .../pir/pir_rel47/vms directory. The UH PIR Gopher server is also updated with the PIR 47 files. Point your gopher to gopher.bchs.uh.edu, select "Fast PIR Lookup" The gopher now includes files from the NRL-3D database. The NRL_3D database did not change between release 46 and release 47. GOPHER LINK INFORMATION Name=PIR Archive, University of Houston Type=1 Port=70 Path= Host=gopher.bchs.uh.edu URL Form: gopher://gopher.bchs.uh.edu dan davison davison@uh.edu -- dr. dan davison/dept. of biochemical and biophysical sciences/univ. of Houston/4800 Calhoun/Houston,TX 77204-5934/davison@uh.edu/DAVISON@UHOU Disclaimer: As always, I speak only for myself, and, usually, only to myself. From owner-proteins@net.bio.net Sun Jan 07 22:00:00 1996 Path: biosci!bcm.tmc.edu!news.tamu.edu!news From: davesmith@bioch.tamu.edu Newsgroups: bionet.molbio.methds-reagnts,bionet.molbio.proteins Subject: Re: fresh transformations for protein expression?? Date: Mon, 08 Jan 1996 21:20:05 GMT Organization: Texas A&M University, College Station, TX Lines: 50 Message-ID: <4crqmo$fhc@news.tamu.edu> References: <4ch87s$15oc@majestix.uni-muenster.de> NNTP-Posting-Host: 486young1.tamu.edu X-Newsreader: Forte Free Agent 1.0.82 Xref: biosci bionet.molbio.methds-reagnts:38370 bionet.molbio.proteins:6649 Torsten Boerchers wrote: >Dear colleagues >for quite some time we are successfully overexpressing >recombinant proteins in bacteria,mostly with the pET >system and mostly simple, nice, soluble proteins. >The more we were surprised that we now on two occacions >with similar proteins encountered problems (i.e. no >expression) which were solved by freshly transforming >the E.coli (BL31(DE3)) with the expression vector instead >of starting from a plate. So fine, that works, but I would >nevertheless like to know whether others had similar >experiences and whether there is an explanation for this >behaviour available. I heared from some other folks routinely >doing fresh transformations but could not get a rationale. >Looking forward to some ideas >Regards >Torsten Torsten, We routinely use BL21 (DE3) harboring various constructs, all of which are pET11a derivatives of one sort or another. All of our expressions with this system to date have been membrane proteins (lysis proteins from bacteriophage). We have seen the effect you mentioned consistently since first using the system. We rationalize this effect to 1) the toxicity or nature of the protein being expressed and 2) the dual layer of transcriptional control (repression at the promoter for T7 RNAP and at the hybrid promoter on the vector), although much tighter than standard cloning and expression vectors, IS LEAKY. In other words, in our experience, E. coli has difficulty with membrane protein overproduction, and the pET system is never OFF. Thus, any colonies on a plate are almost guaranteed to have a low background level of plasmid-encoded protein. Further complications are due to plasmid copy number effects....a fresh transformant will have FEWER plasmids per cell and presumably lower background levels of protein expression as compared to an aged cell on a plate where copy number is maximized due to stationary growth phase effects. Is anyone aware of other problems which we haven't considered? Does anyone have any suggestions on how to alleviate the need to use fresh transformants for every experiment? I'd sure like life to be easier! Dave Smith From owner-proteins@net.bio.net Mon Jan 08 22:00:00 1996 Path: biosci!bcm.tmc.edu!cs.utexas.edu!howland.reston.ans.net!gatech!newsfeed.internetmci.com!in1.uu.net!fdn.fr!jussieu.fr!unilim.fr!news From: Maftah Newsgroups: bionet.molbio.proteins Subject: Collagenase ...Help !!! Date: 9 Jan 1996 15:08:26 GMT Organization: Laboratoire de Biotechnologie Lines: 21 Message-ID: <4cu0db$sk2@sunncs.unilim.fr> NNTP-Posting-Host: 164.81.110.8 Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 1.1N (Macintosh; I; PPC) X-URL: news:bionet.molbio.proteins Dear netters, Does anybody know the required conditions for collagenase activity ? I'm trying to use collagenase with fusion peptides, and would like to split 34 amino acid peptides at position 26, leaving an octapeptide from the C terminal end (collagenase is believed to split the peptide bond between aa 26 and 27). Thanks a lot to answer to : Abdou Maftah institut de Biotechnologie 123, Avenue Albert Thomas 87060 Limoges cedex France Phone (33) 55 45 76 84 Fax (33) 55 45 76 53 email maftah@unilim.fr From owner-proteins@net.bio.net Mon Jan 08 22:00:00 1996 Path: biosci!bcm.tmc.edu!news.msfc.nasa.gov!newsfeed.internetmci.com!gatech!newsxfer.itd.umich.edu!news.mtu.edu!msunews!harbinger.cc.monash.edu.au!news.mel.connect.com.au!munnari.OZ.AU!metro!metro!unsw.edu.au!usenet From: R James Storer Newsgroups: bionet.molbio.proteins Subject: Sequence Alignment Date: 9 Jan 1996 06:07:21 GMT Organization: The University of New South Wales Faculty of Medicine Lines: 23 Message-ID: <4ct0mp$kd2@mirv.unsw.edu.au> NNTP-Posting-Host: 149.171.66.51 Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 1.2N (Windows; I; 16bit) Does anyone know of any protein sequence alignment program (available on the net). I am looking for a program which uses standard parameters, like the on which uses the clustal algorithm (higgins,D.G.& Sharp,P.M.(1988)GENE 73,237-244.) and also to quantify the similarity between segments like the ALIGN program described in "Methods in Enzymology"Vol.93, Dayhoff,M.O.,Baker,W.C.&Hunt pp.521-545. I use Windows 3.11 in an IBM comp. PC environment and so am looking for something that will run using a DOS or Windows operating system. Want I am aiming to do is to compare recently published protein sequences with one another to compare interspecies/intertissue similarities. Also, if you are feeling really generous, I want to know how to get access to protein sequence databases, such as Protein Information Resource (PIR), and how to compare bits of new sequence, eg. from my N-terminal degradation results, with published sequences to see if they have been previously described or to find possible similarity/homology to other proteins. Please send answer by e-mail, but feel free to post the answer as it is possibly a FAQ. From owner-proteins@net.bio.net Mon Jan 08 22:00:00 1996 Path: biosci!bcm.tmc.edu!news.msfc.nasa.gov!newsfeed.internetmci.com!newsxfer2.itd.umich.edu!newsxfer.itd.umich.edu!news2.acs.oakland.edu!cwis-20.wayne.edu!usenet From: Anton Scott Goustin Newsgroups: bionet.molbio.proteins Subject: Re: Searching Genbank for motifs Date: 9 Jan 1996 19:00:36 GMT Organization: Wayne State University, Detroit, MI USA Lines: 5 Message-ID: <4cue0k$m8@cwis-20.wayne.edu> References: NNTP-Posting-Host: asgoff.biosci.wayne.edu Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 1.1N (Macintosh; I; PPC) To: vurquidiI@molbiol.ox.ac.uk,dcvitkov@dental.ufl.edu, X-URL: news:vurquidiI.1171506332A@news.ox.ac.uk Try this WWW site: http://dot.imgen.bcm.tmc.edu:9331/seq-search/Help/beauty.html From owner-proteins@net.bio.net Mon Jan 08 22:00:00 1996 Path: biosci!bcm.tmc.edu!news.msfc.nasa.gov!newsfeed.internetmci.com!uwm.edu!msunews!netnews.upenn.edu!mail.med.upenn.edu!wellsg From: wellsg@mail.med.upenn.edu (Gregg B Wells) Newsgroups: bionet.molbio.proteins,bionet.software Subject: Re: WWW seq alignment site with "jumbling" test of significance? Followup-To: bionet.molbio.proteins,bionet.software Date: 10 Jan 1996 03:13:36 GMT Organization: University of Pennsylvania Lines: 55 Message-ID: <4cvat0$mf9@netnews.upenn.edu> References: NNTP-Posting-Host: mail.med.upenn.edu X-Newsreader: TIN [version 1.2 PL2-upenn1.1] Xref: biosci bionet.molbio.proteins:6661 bionet.software:14393 Neil Clarke (Neil.Clarke@qmail.bs.jhu.edu) wrote: : Anyone know of a WWW site that does an alignment between two protein : sequences with the option of rerunning the alignement using a randomized : ("jumbled") sequence? as a test of significance? Having just given a : lecture on sequence analysis during which I extolled the virtues of the : Web, I was surprised to find that I couldn't find a good old-fashioned : Needleman-Wunsch type program out there. Lots of local homology search : algorithms and dynamic programming algorithms for database searching, but : no 'optimal' sequence alignment stuff. Help, please! _______________________________________________________________________ The Smith-Waterman algorithm can be used at the GenQuest site from Oak Ridge National Laboratory for searching protein and DNA sequences. Here is the email address for more information: ---------------------------------- GENQUEST E-MAIL SERVER USER MANUAL ---------------------------------- GenQuest can be accessed by sending e-mail to: Q@ornl.gov Messages to GENQUEST begin with a set of keywords which specify the options to be used in the search. Two key words are mandatory: TYPE and SEQ. The remainder are optional or have default settings. GENQUEST is case insensitive. EXAMPLE of a typical query: TYPE DNA6 TARGET SwissProt METHOD SW -g 13 MATRIX PAM120 SCORE 50 ALIGN 20 SEQ ATCTATCGTCGAGCTGGTGTCTGTGCTAGTCCACAGACAGHCTCGCTATATATGCT CGTTTTAAAGCTCGTATATATGCTCTCGCTAGTCCGATCGATGCTCGATCGCTAGTA TCGTATGATTCTTG END This example translates the given DNA sequence in 6 frames and searches SwissProt, using Smith-Waterman with gap penalty of 13, PAM120 matrix, and showing top 50 matches and top 20 alignments. Gregg Wells Department of Pathology University of Pennsylvania Philadelphia, Pennsylvania USA email: wells@athens.dental.upenn.edu From owner-proteins@net.bio.net Mon Jan 08 22:00:00 1996 Newsgroups: bionet.molbio.methds-reagnts,bionet.molbio.proteins Path: biosci!bcm.tmc.edu!news.msfc.nasa.gov!newsfeed.internetmci.com!howland.reston.ans.net!torn!utnut!oci!news From: Flip Hoedemaeker Subject: Re: fresh transformations for protein expression?? Content-Type: text/plain; charset=us-ascii Message-ID: <30F2E363.41C6@oci.utoronto.ca> Sender: news@oci.utoronto.ca Content-Transfer-Encoding: 7bit Organization: Ontario Cancer Institute - U of Toronto References: <4ch87s$15oc@majestix.uni-muenster.de> <4crqmo$fhc@news.tamu.edu> Mime-Version: 1.0 Date: Tue, 9 Jan 1996 21:51:31 GMT X-Mailer: Mozilla 2.0b2 (X11; I; IRIX 5.3 IP20) Lines: 32 Xref: biosci bionet.molbio.methds-reagnts:38432 bionet.molbio.proteins:6660 davesmith@bioch.tamu.edu wrote: > Torsten, > We routinely use BL21 (DE3) harboring various constructs, all of which > are pET11a derivatives of one sort or another. All of our expressions > with this system to date have been membrane proteins (lysis proteins > from bacteriophage). We have seen the effect you mentioned > consistently since first using the system. We rationalize this effect > to 1) the toxicity or nature of the protein being expressed and 2) the > dual layer of transcriptional control (repression at the promoter for > T7 RNAP and at the hybrid promoter on the vector), although much > tighter than standard cloning and expression vectors, IS LEAKY. In > other words, in our experience, E. coli has difficulty with membrane > protein overproduction, and the pET system is never OFF. Thus, any > colonies on a plate are almost guaranteed to have a low background > level of plasmid-encoded protein. [..] > Is anyone aware of other problems which we haven't considered? > Does anyone have any suggestions on how to alleviate the need to use > fresh transformants for every experiment? I'd sure like life to be > easier! > > Dave Smith If you think leaky expression is the problem in your case, you should try the pLysS variant of BL21(DE3). The small amount of T7 lysozyme produced inhibits T7 RNA polymerase, decreasing leaky expression. If this doesn't work, try pLysE, which makes more lysozyme. Alternatively, try other (DE3) hosts, some people claim they can better control expression in e.g. HMS 714 (DE3) Hope this helps, Flip From owner-proteins@net.bio.net Mon Jan 08 22:00:00 1996 Path: biosci!bcm.tmc.edu!news.msfc.nasa.gov!elroy.jpl.nasa.gov!swrinde!newsfeed.internetmci.com!in2.uu.net!van-bc!unixg.ubc.ca!news From: Tony Fu Newsgroups: bionet.molbio.proteins Subject: Macromolecular Crystal Packing in Virtual Reality Date: 9 Jan 1996 23:03:46 GMT Organization: University of British Columbia Lines: 29 Message-ID: <4cus8i$7eb@nntp.ucs.ubc.ca> NNTP-Posting-Host: xray1.chem.ubc.ca Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 1.1N (X11; I; IRIX 5.3 IP20) X-URL: news:bionet.molbio.proteins Dear folks, We would like to announce our WWW server on Macromolecular Crystal Packing in Virtual Reality at http://laue.biochem.ubc.ca:8080/cgi-bin/ssis/banff/xpack.html It allows easy 3D visualization of PDB crystal packing in real time. All you needed is a VRML browser and a PDB file accessible by URL (ftp or http). Have fun! Tony -- =================================================================== Tai Y. Fu (Tony) ................................ Department of Biochemistry & Molecular Biology Protein Engineering Network of Centres of Excellence University of British Columbia 2146 Health Sciences Mall, Vancouver BC, CANADA V6T 1Z3. tel: (604)-822-5007 fax: (604)-822-5227 email: tony@laue.biochem.ubc.ca http://laue.biochem.ubc.ca:8080/tony.html =================================================================== From owner-proteins@net.bio.net Mon Jan 08 22:00:00 1996 Newsgroups: bionet.molbio.methds-reagnts,bionet.molbio.proteins Path: biosci!bcm.tmc.edu!news.msfc.nasa.gov!newsfeed.internetmci.com!tank.news.pipex.net!pipex!peer-news.britain.eu.net!newsfeed.ed.ac.uk!leeds.ac.uk!news From: bmbckh@biovax.leeds.ac.uk (Dr. Arthur L. Kruckeberg) Subject: Re: fresh transformations for protein expression?? Originator: -Not-Authenticated-[9535]@leeds.ac.uk Message-ID: <1996Jan9.155945.7422@leeds.ac.uk> NNTP-Posting-Host: bmbmac01.leeds.ac.uk Xdisclaimer: No attempt was made to authenticate the sender's name. Organization: Department of Biochemistry and Molecular Biology X-Posted-From: InterNews 1.0@gps.leeds.ac.uk. Date: Tue, 9 Jan 1996 15:59:44 +0000 (GMT) References: <4ch87s$15oc@majestix.uni-muenster.de> <4crqmo$fhc@news.tamu.edu> Lines: 24 Xref: biosci bionet.molbio.methds-reagnts:38408 bionet.molbio.proteins:6655 Hi The problem of non-reproducible overexpression using the same clone stored as a stock in glycerol maybe related to instability of the DNA seqence inserted into the vector. Insert instability can be associated with the RecA phenotype. Since BL21's are RecA+, it maybe advisable to use a RecA- strain or RecA- variant of BL21. Novagen sell a RecA variant of BL21 which they call BLR. Novagen's tel. no. is 800 526 7319. Hope it helps Good luck. Chris ------------------------------------------------------ a.k.a. Chris Hoyle Department of Biochemistry and Molecular Biology University of Leeds phone +44 0113 2333172 Leeds LS2 9JT FAX +44 0113 2333167 Great Britain e-mail bmbckh@biovax.leeds.ac.uk ------------------------------------------------------ From owner-proteins@net.bio.net Mon Jan 08 22:00:00 1996 Path: biosci!bcm.tmc.edu!news.msfc.nasa.gov!newsfeed.internetmci.com!newsxfer2.itd.umich.edu!newsxfer.itd.umich.edu!news2.acs.oakland.edu!cwis-20.wayne.edu!usenet From: Anton Scott Goustin Newsgroups: bionet.molbio.proteins Subject: Re: Searching Genbank for motifs Date: 9 Jan 1996 19:00:51 GMT Organization: Wayne State University, Detroit, MI USA Lines: 5 Message-ID: <4cue13$m8@cwis-20.wayne.edu> References: NNTP-Posting-Host: asgoff.biosci.wayne.edu Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 1.1N (Macintosh; I; PPC) To: vurquidiI@molbiol.ox.ac.uk,dcvitkov@dental.ufl.edu X-URL: news:vurquidiI.1171506332A@news.ox.ac.uk Try this WWW site: http://dot.imgen.bcm.tmc.edu:9331/seq-search/Help/beauty.html From owner-proteins@net.bio.net Mon Jan 08 22:00:00 1996 Path: biosci!bcm.tmc.edu!news.msfc.nasa.gov!newsfeed.internetmci.com!swrinde!cs.utexas.edu!geraldo.cc.utexas.edu!netnews.uthscsa.edu!usenet From: Jeff Seale Newsgroups: bionet.molecules.peptides,bionet.molbio.proteins Subject: prolyl isomerase substrate availability Date: 9 Jan 1996 14:37:54 GMT Organization: University of Texas Health Science Center at San Antonio Lines: 12 Message-ID: <4ctuk2$bdk@cosmos.uthscsa.edu> NNTP-Posting-Host: horowitz_lab1.uthscsa.edu Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 1.2N (Windows; I; 16bit) Xref: biosci bionet.molecules.peptides:166 bionet.molbio.proteins:6653 Is the peptidyl prolyl isomerase substrate H-Ala-Ala-Pro-Tyr(m-NO2)-Ala-NH2 as described in Garcia-Echeverria, et al. BBRC 191, 70-75 (1993) commercially available? Thanks, Jeff Dept. of Biochemistry UTHSC - San Antonio email seale@bioc02.uthscsa.edu From owner-proteins@net.bio.net Mon Jan 08 22:00:00 1996 Path: biosci!GUARANY.CPD.UNB.BR!wagnerf From: wagnerf@GUARANY.CPD.UNB.BR (wagner fontes) Newsgroups: bionet.molbio.proteins Subject: inespecific cleavages Date: 9 Jan 1996 08:26:28 -0800 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 10 Sender: daemon@net.bio.net Distribution: world Message-ID: NNTP-Posting-Host: net.bio.net I'm doing chemical cleavages on a 55k protein with Iodosobenzoic acid and with Cyanogen bromide. The problem is that these cleavages are supposed to be very specific, but i am obtaining very inespecific cleavages. Does anybody know why could it happen, or any references about the same situation ? P.S. The reagents are new, the method was performed as described by the authors and the results are reproducible. Thanks, Wagner. From owner-proteins@net.bio.net Tue Jan 09 22:00:00 1996 Path: biosci!bcm.tmc.edu!pendragon!news.msfc.nasa.gov!newsfeed.internetmci.com!vixen.cso.uiuc.edu!koniskyj3.life.uiuc.edu!user From: Dan_Ferber@qms1.life.uiuc.edu (Dan Ferber) Newsgroups: bionet.molbio.proteins Subject: SDS Date: Wed, 10 Jan 1996 10:00:31 -0600 Organization: University of Illinois, Dept of Microbiology Lines: 7 Message-ID: NNTP-Posting-Host: koniskyj3.life.uiuc.edu Could anyone please direct me to references that deal with the interference of negatively charged amino acid residues on the interaction of sodium dodecyl sulphate with a denatured protein, i.e. in an SDS-PAGE gel. Thanks Marc Stees From owner-proteins@net.bio.net Tue Jan 09 22:00:00 1996 Path: biosci!bcm.tmc.edu!news.msfc.nasa.gov!newsfeed.internetmci.com!EU.net!Austria.EU.net!newsfeed.ACO.net!swidir.switch.ch!swsbe6.switch.ch!scsing.switch.ch!news.belwue.de!fu-berlin.de!news.dfn.de!gs.dfn.de!uni-erlangen.de!winx03!wpxx02.toxi.uni-wuerzburg.de!not-for-mail From: krasel@wpxx02.toxi.uni-wuerzburg.de (Cornelius Krasel) Newsgroups: bionet.molbio.methds-reagnts,bionet.molbio.proteins Subject: Re: fresh transformations for protein expression?? Date: 10 Jan 1996 09:53:57 GMT Organization: University of Wuerzburg, Germany Lines: 16 Message-ID: <4d02bl$t8l@winx03.informatik.uni-wuerzburg.de> References: <4ch87s$15oc@majestix.uni-muenster.de> <4crqmo$fhc@news.tamu.edu> <4cscqf$h2n@winx03.informatik.uni-wuerzburg.de> NNTP-Posting-Host: wpxx02.toxi.uni-wuerzburg.de X-Newsreader: TIN [UNIX 1.3 950824BETA PL0] Xref: biosci bionet.molbio.methds-reagnts:38451 bionet.molbio.proteins:6662 I made a mistake in my previous post :-( Cornelius Krasel (krasel@wpxx02.toxi.uni-wuerzburg.de) wrote: > If you cotransform BL21(DE3) with pLysS or pLysL the system becomes less ^^^^^pLysE > leaky. BL21(DE3)pLysS are available from several companies; I don't know > about BL21(DE3)pLysL (which is a stronger repressor than pLysS). ^^^^^pLysE Sorry, --Cornelius. -- /* Cornelius Krasel, U Wuerzburg, Dept. of Pharmacology, Versbacher Str. 9 */ /* D-97078 Wuerzburg, Germany email: phak004@rzbox.uni-wuerzburg.de SP3 */ /* "Science is the game we play with God to find out what His rules are." */ From owner-proteins@net.bio.net Tue Jan 09 22:00:00 1996 Path: biosci!bcm.tmc.edu!news.msfc.nasa.gov!elroy.jpl.nasa.gov!swrinde!howland.reston.ans.net!newsfeed.internetmci.com!in1.uu.net!news.ultranet.com!mikes-mac.ultranet.com!user From: mtyo@ultranet.com (Mike Tyo) Newsgroups: bionet.molbio.proteins Subject: Boston area Fermentation Group Organizational Meeting Date: Wed, 10 Jan 1996 13:03:28 -0500 Organization: UltraNet Communications, Inc. Lines: 16 Message-ID: NNTP-Posting-Host: mikes-mac.ultranet.com To all Boston area biotech scientists: A new group is forming in the Boston area, composed of scientists and engineers interested in recombinant protein expression, production and purification. We are not (yet) affiliated with any other professional organization. On Thursday January 18 at 7:00 pm, we are holding our first organizational dinner meeting at Joyce Chen's Restaurant across Route 2 from Alewife MBTA station. We plan to discuss the nature of the group, its name, where and when to meet, and a format for future scientific presentations. All those interested should contact me at my email address (mtyo@ultranet.com) or work number (AutoImmune, Inc. 617-860-0710) or so I can make dinner reservations. I hope to see you there! Readers of this message should feel free to repost it in any other suitable place. --Michael Tyo From owner-proteins@net.bio.net Tue Jan 09 22:00:00 1996 Path: biosci!bcm.tmc.edu!news.msfc.nasa.gov!newsfeed.internetmci.com!gatech!sdd.hp.com!vixen.cso.uiuc.edu!howland.reston.ans.net!nntp.coast.net!swidir.switch.ch!serra.unipi.it!t500.vol.it!news From: Paolo Tomasi MD PhD Newsgroups: bionet.molbio.proteins,bionet.software Subject: Re: DOS/Windows sequence software? Date: Wed, 10 Jan 1996 02:40:58 +0100 Organization: Ospedale di Olbia Lines: 20 Message-ID: <30F3192A.D2C@mbox.vol.it> References: <4cna0c$nrc@usenet.ucs.indiana.edu> NNTP-Posting-Host: volss2.vol.it Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 2.0b5 (Win95; I) Xref: biosci bionet.molbio.proteins:6670 bionet.software:14407 There's also a program called SeqAid II that helps with point 5 (i.e. protein structure "guesses"). It's DOS, but quite good. To find the FTP server, look in the Biologic Software section of http://www.gdb.org/ Paolo Tomasi In article , Andy Bates wrote: > It needs to be: > >1. DOS/Windows >2. free >3. able to find restriction sites and search for short strings with >mismatches/gaps >4. able to find ORFs and translate them >5. hopefully able to give some details of the hypothetical proteins, >e.g. mol wt., amino acid composition. From owner-proteins@net.bio.net Tue Jan 09 22:00:00 1996 Path: biosci!bcm.tmc.edu!news.msfc.nasa.gov!newsfeed.internetmci.com!in1.uu.net!EU.net!Austria.EU.net!newsfeed.ACO.net!swidir.switch.ch!scsing.switch.ch!news.belwue.de!fu-berlin.de!zrz.TU-Berlin.DE!news.dfn.de!gs.dfn.de!uni-erlangen.de!winx03!wpxx02.toxi.uni-wuerzburg.de!not-for-mail From: krasel@wpxx02.toxi.uni-wuerzburg.de (Cornelius Krasel) Newsgroups: bionet.molbio.proteins Subject: Re: Sequence Alignment Date: 10 Jan 1996 10:01:03 GMT Organization: University of Wuerzburg, Germany Lines: 46 Message-ID: <4d02ov$t8l@winx03.informatik.uni-wuerzburg.de> References: <4ct0mp$kd2@mirv.unsw.edu.au> NNTP-Posting-Host: wpxx02.toxi.uni-wuerzburg.de X-Newsreader: TIN [UNIX 1.3 950824BETA PL0] R James Storer (rjstorer@acsusun.acsu.unsw.edu.au) wrote: > I use Windows 3.11 in an IBM comp. PC environment and so am looking for > something that will run using a DOS or Windows operating system. I *think* Clustal is available for DOS. There are some other programs you might want to play with, such as Macaw (for Windows). > Also, if you are feeling really generous, I want to know how to get > access to protein sequence databases, such as Protein Information > Resource (PIR), and how to compare bits of new sequence, eg. from my > N-terminal degradation results, with published sequences to see if they > have been previously described or to find possible similarity/homology to > other proteins. Check out some of the following servers: - GENBANK, SWISSPROT, PIR, TFD (Transcription Factor database) and PROSITE http://www.nih.gov/molbio - PIR is also available at the Martinsrieder Institut fuer Proteinsequenzen http://www.mips.biochem.mpg.de/ (probably very slow for you since you are Australia-based) - The European Bioinformatics Institute (EBI) at Cambridge (UK) offers SRS, a sequence retrieval system which indexes all of the major databases. http://www.ebi.ac.uk/srs/srsc - ExPASy server at Geneva carries SWISSPROT and PROSITE http://expasy.hcuge.ch/ - John Hopkins Prot-Web server carries OWL (another non-redundant protein sequence database), NRL-3D and PIR http://www.gdb.org/ - BLAST searches at the NCBI http://www.ncbi.nlm.nih.gov/Recipon/index.html You might also be interested in Entrez. --Cornelius. -- /* Cornelius Krasel, U Wuerzburg, Dept. of Pharmacology, Versbacher Str. 9 */ /* D-97078 Wuerzburg, Germany email: phak004@rzbox.uni-wuerzburg.de SP3 */ /* "Science is the game we play with God to find out what His rules are." */ From owner-proteins@net.bio.net Tue Jan 09 22:00:00 1996 Path: biosci!bcm.tmc.edu!news.msfc.nasa.gov!newsfeed.internetmci.com!gatech!sdd.hp.com!vixen.cso.uiuc.edu!howland.reston.ans.net!nntp.coast.net!swidir.switch.ch!serra.unipi.it!t500.vol.it!news From: Paolo Tomasi MD PhD Newsgroups: bionet.molbio.proteins,bionet.software Subject: Re: DOS/Windows sequence software? Date: Wed, 10 Jan 1996 02:38:59 +0100 Organization: Ospedale di Olbia Lines: 20 Message-ID: <30F318B3.781F@mbox.vol.it> References: <4cna0c$nrc@usenet.ucs.indiana.edu> NNTP-Posting-Host: volss2.vol.it Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 2.0b5 (Win95; I) Xref: biosci bionet.molbio.proteins:6669 bionet.software:14406 There's also a program called SeqAid II that helps with point 5 (i.e. protein structure "guesses"). It's DOS, but quite good. To find the FTP server, look in the Biologic Software section of http://www.gdb.org/ Paolo Tomasi In article , Andy Bates wrote: > It needs to be: > >1. DOS/Windows >2. free >3. able to find restriction sites and search for short strings with >mismatches/gaps >4. able to find ORFs and translate them >5. hopefully able to give some details of the hypothetical proteins, >e.g. mol wt., amino acid composition. From owner-proteins@net.bio.net Tue Jan 09 22:00:00 1996 Path: biosci!bcm.tmc.edu!news.msfc.nasa.gov!newsfeed.internetmci.com!howland.reston.ans.net!lamarck.sura.net!news.uky.edu!news From: Michael Thompson Newsgroups: bionet.molbio.proteins Subject: Re: Pretty ashamed to post this... Date: 10 Jan 1996 20:25:24 GMT Organization: University of Kentucky Lines: 23 Message-ID: <4d17bk$pg6@service2.uky.edu> References: <4c1qh3$gkl@server-b.cs.interbusiness.it> <4c6f2f$3ac@news.jhu.edu> <4c6uqc$9r3@server-b.cs.interbusiness.it> NNTP-Posting-Host: 128.163.70.124 Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 1.12(Macintosh; I; PPC) X-URL: news:4c6uqc$9r3@server-b.cs.interbusiness.it There are several mechanism by which proteins may be diverted to other fates. Proteins destined to reside at the ER membrane often contain a dilysine motif at the C-terminus (KKXX) which causes the protein to be continuously recycled back to the ER membrane. Soluble ER proteins may contain an HDEL sequence at the C-terminus. Proteins destined for the lysosome may have an SKL sequence at the C-terminus. There are also sequences which may cause a protein to be targetted to the nucleus. This is only one way in which protein targeting has been shown to be achieved; it is likely that other mechanisms will be discovered. And, of course, there are many more targeting sequences known than I have posted here. Hope this was of help to you Michael Thompson University of Kentucky Dept. of Biochemistry Lexington, KY 40536-0084 -- Where am I going and why am I in this handbasket? From owner-proteins@net.bio.net Tue Jan 09 22:00:00 1996 Path: biosci!bloom-beacon.mit.edu!gatech!sdd.hp.com!vixen.cso.uiuc.edu!howland.reston.ans.net!nntp.coast.net!swidir.switch.ch!serra.unipi.it!t500.vol.it!news From: Paolo Tomasi MD PhD Newsgroups: bionet.molbio.proteins,bionet.software Subject: Re: DOS/Windows sequence software? Date: Wed, 10 Jan 1996 02:30:27 +0100 Organization: Ospedale di Olbia Lines: 6 Message-ID: <30F316B3.440F@mbox.vol.it> References: <4cna0c$nrc@usenet.ucs.indiana.edu> NNTP-Posting-Host: volss2.vol.it Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 2.0b5 (Win95; I) Xref: biosci bionet.molbio.proteins:6673 bionet.software:14412 There's also a program called SeqAid II that helps with point 5 (i.e. protein structure "guesses"). It's DOS, but quite good. To find the FTP server, look in the Biologic Software section of http://www.gdb.org/ Paolo Tomasi From owner-proteins@net.bio.net Tue Jan 09 22:00:00 1996 Path: biosci!bcm.tmc.edu!news.msfc.nasa.gov!newsfeed.internetmci.com!gatech!sdd.hp.com!vixen.cso.uiuc.edu!howland.reston.ans.net!nntp.coast.net!swidir.switch.ch!serra.unipi.it!t500.vol.it!news From: Paolo Tomasi MD PhD Newsgroups: bionet.molbio.proteins,bionet.software Subject: Re: DOS/Windows sequence software? Date: Wed, 10 Jan 1996 02:48:15 +0100 Organization: Ospedale di Olbia Lines: 6 Message-ID: <30F31ADF.1D76@mbox.vol.it> References: <4cna0c$nrc@usenet.ucs.indiana.edu> NNTP-Posting-Host: volss2.vol.it Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 2.0b5 (Win95; I) Xref: biosci bionet.molbio.proteins:6672 bionet.software:14409 There's also a program called SeqAid II that helps with point 5 (i.e. protein structure "guesses"). It's DOS, but quite good. To find the FTP server, look in the Biologic Software section of http://www.gdb.org/ Paolo Tomasi From owner-proteins@net.bio.net Tue Jan 09 22:00:00 1996 Path: biosci!bcm.tmc.edu!news.msfc.nasa.gov!newsfeed.internetmci.com!gatech!sdd.hp.com!vixen.cso.uiuc.edu!howland.reston.ans.net!nntp.coast.net!swidir.switch.ch!serra.unipi.it!t500.vol.it!news From: Paolo Tomasi MD PhD Newsgroups: bionet.molbio.proteins,bionet.software Subject: Re: DOS/Windows sequence software? Date: Wed, 10 Jan 1996 02:33:22 +0100 Organization: Ospedale di Olbia Lines: 6 Message-ID: <30F31762.2C5F@mbox.vol.it> References: <4cna0c$nrc@usenet.ucs.indiana.edu> NNTP-Posting-Host: volss2.vol.it Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 2.0b5 (Win95; I) Xref: biosci bionet.molbio.proteins:6671 bionet.software:14408 There's also a program called SeqAid II that helps with point 5 (i.e. protein structure "guesses"). It's DOS, but quite good. To find the FTP server, look in the Biologic Software section of http://www.gdb.org/ Paolo Tomasi From owner-proteins@net.bio.net Tue Jan 09 22:00:00 1996 Path: biosci!bcm.tmc.edu!pendragon!news.msfc.nasa.gov!newsfeed.internetmci.com!globe.indirect.com!usenet From: coburn@indirect.com (anne coburn) Newsgroups: bionet.molbio.proteins Subject: PROTEIN CHEMIST OPENING Date: 10 Jan 1996 21:18:19 GMT Organization: Coburn Scientific Lines: 39 Sender: -Not-Authenticated-[3112] Message-ID: <4d1aer$pri@globe.indirect.com> NNTP-Posting-Host: s41.tucslip.indirect.com X-Posted-From: InterNews 1.0.7@s41.tucslip.indirect.com Xdisclaimer: No attempt was made to authenticate the sender's name. A leading biotechnology company in Southern California seeks a: RESEARCH ASSOCIATE II/III - PROTEIN CHEMISTRY This company is at the forefront in the discovery, expression, purification, characterization and laboratory-scale development of new protein therapeutics. 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For immediate and confidential consideration, contact ANNE COBURN Coburn Search & Consulting 4449 East Whitman Tucson, AZ 85711-3515 Phone: 520-881-0084 Fax: 520-321-1475 e-mail: coburn@indirect.com From owner-proteins@net.bio.net Tue Jan 09 22:00:00 1996 Path: biosci!bcm.tmc.edu!news.msfc.nasa.gov!newsfeed.internetmci.com!gatech!news-feed-1.peachnet.edu!news.cc.emory.edu!news.cc.emory.edu!not-for-mail From: jsoria@larry.cc.emory.edu (Jennie P Soria) Newsgroups: bionet.molbio.proteins Subject: tubulin Date: 10 Jan 1996 17:07:59 -0500 Organization: Emory University Lines: 5 Message-ID: <4d1dbv$e7g@larry.cc.emory.edu> NNTP-Posting-Host: larry.cc.emory.edu X-Newsreader: TIN [version 1.2 PL2] does anyone know if there exists a general expression system for beta1,2,3 tubulin expression ? jps From owner-proteins@net.bio.net Wed Jan 10 22:00:00 1996 Path: biosci!bcm.tmc.edu!pendragon!news.msfc.nasa.gov!elroy.jpl.nasa.gov!swrinde!tank.news.pipex.net!pipex!sunsite.doc.ic.ac.uk!sunews!suma3!afrrobjs From: "Simone J. Robinson" Newsgroups: bionet.molbio.proteins Subject: pET 3d Date: Thu, 11 Jan 1996 16:55:15 +0000 Organization: University of Reading, U.K. Lines: 11 Message-ID: NNTP-Posting-Host: suma3-e2.reading.ac.uk Mime-Version: 1.0 Content-Type: TEXT/PLAIN; charset=US-ASCII Hello, We are about to start working with the plasmid pET 3d. Is there anybody working with this plasmid who may be able to offer any advice or any references that may be appropriate. Many thanks in advance. Simone Robinson From owner-proteins@net.bio.net Wed Jan 10 22:00:00 1996 Path: biosci!bcm.tmc.edu!news.msfc.nasa.gov!newsfeed.internetmci.com!in1.uu.net!EU.net!Norway.EU.net!nntp.uio.no!news.kth.se!newsfeed.sunet.se!news01.sunet.se!sunic!news99.sunet.se!news.uni-c.dk!news From: vadtsvet@biochem.ou.dk (Vadim Tsvetnitsky) Newsgroups: bionet.molbio.proteins Subject: His-tagged proteins Date: 11 Jan 1996 10:27:30 GMT Organization: Odense University, DENMARK Lines: 10 Message-ID: <4d2omi$ej7@news.uni-c.dk> NNTP-Posting-Host: vadim.biochem.ou.dk X-Newsreader: WinVN 0.92.6+ Hi, Has anyone tried to regenerate Ni-NTA agarose used for purification of his-tagged proteins? I understand from the broushure that a particular batch can only be used 3 to 5 times for the purification of one protein and should be trashed. Is it so? Thanks. Vadim Tsvetnitsky vadtsvet@biochem.ou.dk From owner-proteins@net.bio.net Wed Jan 10 22:00:00 1996 Path: biosci!bcm.tmc.edu!cs.utexas.edu!howland.reston.ans.net!EU.net!Norway.EU.net!nntp-oslo.UNINETT.no!nntp-trd.UNINETT.no!newsfeed.sunet.se!news01.sunet.se!sunic!news99.sunet.se!columba.udac.uu.se!image.bmc.uu.se!user From: msz@bio.embnet.se (Michael Szardenings) Newsgroups: bionet.molbio.methds-reagnts,bionet.molbio.proteins Subject: Re: fresh transformations for protein expression?? Date: Thu, 11 Jan 1996 13:31:14 +0100 Organization: Uppsala University, BMC, Farm.Farm. Lines: 52 Message-ID: References: <4ch87s$15oc@majestix.uni-muenster.de> NNTP-Posting-Host: image.bmc.uu.se Xref: biosci bionet.molbio.methds-reagnts:38513 bionet.molbio.proteins:6675 In article <4ch87s$15oc@majestix.uni-muenster.de>, Torsten Boerchers wrote: > The more we were surprised that we now on two occacions > with similar proteins encountered problems (i.e. no > expression) which were solved by freshly transforming > the E.coli (BL31(DE3)) with the expression vector instead > of starting from a plate. Dear Torsten, this problem is indeed very common as the many replies on the list show. I would like to add (from my own history) a few points: 1. This is no problem that is specific for BL21, it is indeed common to all systems where the expression of the foreign gene is not properly regulated. In case of BL21/pET I would recommend: a) use media with 1-2% glucose (keeps lac-promoter down) b) use pLysE (as others already told) c) NEVER grow precultures or plates to the stationary growth phase, then the cells are most sensitive to heterologous expression of toxic genes. d) use 30 deg C or lower for cultivation. e) check the uninduced cultures, I once saw almost as much protein in these as in the induced cells (return from here to 1a...) 2. Many people are claiming, that these effects are due to plasmid instability. I have never seen any real proof for this, i.e. effects that go beyond the normal loss of plasmids due to selection for non-expressing clones. In the worst case I have seen growth arrest of the culture followed some time later by rapid growing cells without plasmid, as the ampicillin used in the experiment had been degraded during the initial growth. Another effect has been, that proteins, that ought to be secreted, were much more sensitive to such effects. This may be due to the fact, that more genes are involved in the proper expression of such proteins. I think that spontanous mutations may result then easier in clones expressing less protein. I have had such cases, were the plasmid from the culture was absolutely o.k., but the expression level was x10E-4 (!) of the level achieved under optimal conditions. Good luck, you may need a bit Michael ========================================================================= * * ****** ******** ********************************************* ** ** *** ** ******** * Michael Szardenings * *** *** ***** *** * Biomedical Center, Pharm. Pharm. * **** **** ***** *** * Box 591 Tel.:int46-18-17422* ** *** ** ** *** ******** * S-751 24 Uppsala FAX :int46-18-55971* ** * ** ****** ******** ********************************************* ========================================================================= INTERNET : MSZ@bmc.uu.se ========================================================================= From owner-proteins@net.bio.net Wed Jan 10 22:00:00 1996 Path: biosci!bcm.tmc.edu!pendragon!news.msfc.nasa.gov!elroy.jpl.nasa.gov!swrinde!gatech!sdd.hp.com!col.hp.com!csn!magnus.acs.ohio-state.edu!lerc.nasa.gov!purdue!news.bu.edu!usenet From: alwang@bu.edu (Allen) Newsgroups: bionet.molbio.proteins Subject: Hyaluronidase Date: Thu, 11 Jan 1996 21:51:22 GMT Organization: Boston University Lines: 5 Message-ID: <4d3ut1$p3m@news.bu.edu> Reply-To: alwang@bu.edu NNTP-Posting-Host: ppp-81-20.bu.edu X-Newsreader: Forte Free Agent 1.0.82 Can anyone send me a 3D rendered picture of hyaluronidase? Also, if you do know, can you give me the chemical composition of hyaluronidase? From owner-proteins@net.bio.net Wed Jan 10 22:00:00 1996 Path: biosci!bcm.tmc.edu!news.msfc.nasa.gov!newsfeed.internetmci.com!vixen.cso.uiuc.edu!howland.reston.ans.net!news-e1a.megaweb.com!newstf01.news.aol.com!newsbf02.news.aol.com!not-for-mail From: cpnintt@aol.com (CPN INTt) Newsgroups: bionet.molbio.proteins Subject: If you are interested in Cellulase and or Xylanase Please Register Here Date: 12 Jan 1996 02:00:14 -0500 Organization: America Online, Inc. (1-800-827-6364) Lines: 7 Sender: root@newsbf02.news.aol.com Message-ID: <4d50tu$3tu@newsbf02.news.aol.com> Reply-To: cpnintt@aol.com (CPN INTt) If you are actively involved with Cellulase and or Xylanase please register here. If you have e.mail address of other active parties we would welcome the addresses. Thanks In Advance MAE From owner-proteins@net.bio.net Wed Jan 10 22:00:00 1996 Newsgroups: bionet.molbio.methds-reagnts,bionet.molbio.proteins Path: biosci!bcm.tmc.edu!cs.utexas.edu!howland.reston.ans.net!newsfeed.internetmci.com!usenet.eel.ufl.edu!warwick!bsmail!zeus!bijgh From: bijgh@zeus.bris.ac.uk (JG. Head) Subject: Re: fresh transformations for protein expression?? Message-ID: Followup-To: bionet.molbio.methds-reagnts,bionet.molbio.proteins Sender: usenet@uns.bris.ac.uk (Usenet news owner) Nntp-Posting-Host: zeus.bris.ac.uk Organization: University of Bristol, England X-Newsreader: TIN [version 1.2 PL2] References: <4ch87s$15oc@majestix.uni-muenster.de> Date: Thu, 11 Jan 1996 15:30:23 GMT Lines: 27 Xref: biosci bionet.molbio.methds-reagnts:38529 bionet.molbio.proteins:6676 Michael Szardenings (msz@bio.embnet.se) wrote: : 2. Many people are claiming, that these effects are due to plasmid : instability. I have never seen any real proof for this, i.e. effects that : go beyond the normal loss of plasmids due to selection for non-expressing : clones. In the worst case I have seen growth arrest of the culture : followed some time later by rapid growing cells without plasmid, as the : ampicillin used in the experiment had been degraded during the initial : growth. I am expressing what I would expect to be non-toxic constructs using the pET system, and have had similar problems. Freshly transformed cells express well but after a week siting in the fridge the cells won't express at all. Even the glycerols I have made lost the ability to express protein. Like Michael I'm not convinced that these problems are due to plasmid instability. I've tried various experiments to test if the plasmid is unstable and have seen no evidence for it at all. But if this loss of expression is not due to plasmid instability, what else could it be? Any ideas? Cheers, Jared -- Jared Head at the Department of Biochemistry, University of Bristol From owner-proteins@net.bio.net Thu Jan 11 22:00:00 1996 Path: biosci!daresbury!nntp-trd.UNINETT.no!nac.no!ifi.uio.no!news.sics.se!newsfeed.sunet.se!news01.sunet.se!sunic!news99.sunet.se!news.uni-c.dk!news.daimi.aau.dk!moffe From: moffe@daimi.aau.dk (Morten Lauritsen) Newsgroups: bionet.molbio.proteins Subject: Basic question Date: 12 Jan 1996 14:40:59 GMT Organization: DAIMI, Computer Science Dept. at Aarhus University Lines: 31 Message-ID: <4d5rtr$jg@krone.daimi.aau.dk> NNTP-Posting-Host: shekel.daimi.aau.dk Hi all. This was the group that sounded as the most appropriate to post these questions to. Hope nobody minds. I need to know the map between codons (triples of nucleotides) to amino acids. The problem is that this is such a trivial bit of knowledge that nobody seems to have cared to put it on a www site, etc. Also, does anybody know the map between the 20-letter coding of the amino acids into the amino acid names? i.e. What is going on here: MVHWTAEEKAAITSVWQKVNVEHDGHDALGRLLIVYPWTQRYFSNFGNLSNSAAVAGNAKVQAHGKKVLS AVGNAISHIDSVKSSLQQLSKIHATELFVDPENFKRFGGVLVIVLGAKLGTAFTPKVQAAWEKFIAVLVD GLSQGYN I know it is the primary structure of a beta globin, but what is the amino acid names? Please reply by email so the newsgroup isn't bothered too much by this. thanks in advance, Morten Lauritsen. -- Morten Lauritsen | Email: moffe@daimi.aau.dk | To me, boxing is like ballet Spobjergvej 38 10 | ---------------------------| except there's no music, 8220 Brabrand | The world owes you nothing.| no choreography, and Phone: 89 44 91 50 | It was here first. | the dancers hit each other. From owner-proteins@net.bio.net Thu Jan 11 22:00:00 1996 Path: biosci!bloom-beacon.mit.edu!newsfeed.internetmci.com!gatech!sdd.hp.com!col.hp.com!csn!carbon!hermes.cair.du.edu!usenet From: dbovee@du.edu (D. Bovee) Newsgroups: bionet.molbio.proteins Subject: Searching for enzyme using NAD(P)H and ADP ---> NAD(P)+ and ATP Date: Sat, 13 Jan 1996 04:14:36 GMT Organization: University of Denver Lines: 19 Message-ID: <4d7c9t$lct@hermes.cair.du.edu> NNTP-Posting-Host: sl-52.ducomm.du.edu Mime-Version: 1.0 Content-Type: text/plain; charset=US-ASCII Content-Transfer-Encoding: 7bit X-Newsreader: Forte Free Agent 1.0.82 I have been researching this topic, without much luck. I am searching for a *single* enzyme which catalyzes a reaction ultimately converting ADP to ATP (presumably using NAD(P)H. I suspected I might find a dehydrogenase to do this trick...but I haven't had any luck as of yet. One of the biosynthetic reactions employs TWO enzymes to do this: glutamine synthetase and glutamine synthase are used in conjunction whose net reaction makes my products from these reactants. However, for the experiment I am setting up, I need to minimize this reaction to a single enzyme... If you have any ideas, or know of such a system, your help would be greatly appreciated! Please copy any replies to email, if possible. ------ David Bovee Dept. of Chemistry University of Denver From owner-proteins@net.bio.net Thu Jan 11 22:00:00 1996 Path: biosci!bcm.tmc.edu!news.msfc.nasa.gov!newsfeed.internetmci.com!bloom-beacon.mit.edu!senator-bedfellow.mit.edu!usenet From: Michael Yamauchi Newsgroups: bionet.molbio.proteins Subject: Re: His-tagged proteins Date: 13 Jan 1996 00:22:36 GMT Organization: MIT Lines: 14 Message-ID: <4d6u0c$j2i@senator-bedfellow.MIT.EDU> References: <4d2omi$ej7@news.uni-c.dk> NNTP-Posting-Host: tabaker.mit.edu Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 1.1N (Macintosh; I; 68K) To: vadtsvet@biochem.ou.dk X-URL: news:4d2omi$ej7@news.uni-c.dk Vadim- I have re-nickelated Ni-NTA-agarose resins with varying success. I believe Qiagen provides a protocol for doing this. If I remember correctly, it involves stripping the resin with EDTA and then passing nickel sulfate over the column. Alternatively, I have also used Ni-NTA columns many more than 5 times and have had pretty good resolution. Since these resins are available for about $5/ml, I would suggest using the resin as long as you still get good resolution and then either replace it (if it is a relatively small volume), or attempting to regenerate it if it is a large volume. From owner-proteins@net.bio.net Thu Jan 11 22:00:00 1996 Newsgroups: bionet.molbio.proteins Path: biosci!bcm.tmc.edu!news.msfc.nasa.gov!newsfeed.internetmci.com!swrinde!howland.reston.ans.net!psinntp!psinntp!psinntp!psinntp!pfizergate!news From: sam simons Subject: Re: Basic question X-Nntp-Posting-Host: simonssp.pfizer.com Content-Type: text/plain; charset=us-ascii Message-ID: Sender: news@pfizer.com (News Admin) Content-Transfer-Encoding: 7bit Organization: Pfizer Inc References: <4d5rtr$jg@krone.daimi.aau.dk> Mime-Version: 1.0 Date: Fri, 12 Jan 1996 20:32:27 GMT X-Mailer: Mozilla 1.1 (Macintosh; U; PPC) X-Url: news:4d5rtr$jg@krone.daimi.aau.dk Lines: 13 Try the Molecular biologists desk reference<. It's chock full of basic info like this. Just in case the hyperlink doesn't work, here's the URL: http://molbio.info.nih.gov/molbio/desk.html Sam (Haven't figured out a fancy signature yet) For the legal minded among you: This message represents my personal opinions and is in no way to be confused with those of Pfizer, Inc. nor any affiliates and subsidiaries owned wholly or in part by Pfizer, Inc. From owner-proteins@net.bio.net Thu Jan 11 22:00:00 1996 Path: biosci!bcm.tmc.edu!news.msfc.nasa.gov!elroy.jpl.nasa.gov!swrinde!howland.reston.ans.net!nntp.coast.net!swidir.switch.ch!scsing.switch.ch!rzunews.unizh.ch!NewsWatcher!user From: zjons@vetbio.unizh.ch (Zophonias O. Jonsson) Newsgroups: bionet.molbio.proteins Subject: Re: PEST score Date: 12 Jan 1996 21:37:54 GMT Organization: Universitat Zurich-Irchel Lines: 23 Message-ID: References: <4d642k$r3c@news.cict.fr> NNTP-Posting-Host: 130.60.120.18 In article <4d642k$r3c@news.cict.fr>, ducommun@opium.ipbs.fr (ducommun bernard) wrote: > Can anyone tell me where I could run a PEST score calculation on my favourite > protein. http address ? The following link provides what you are looking for http://www.biu.icnet.uk/projects/pest (And I bet that your favourit protein has one potential PEST site with a score of 5.11) :-) Zophonias _____________________________________________________________________ Zophonias O. Jonsson Institut fur Veterinarbiochemie Tel: (41-1)-257-54-75 Universitat Zurich-Irchel Fax: (41-1)-362-05-01 Winterthurerstrasse 190 CH-8057 Zurich Switzerland _____________________________________________________________________ From owner-proteins@net.bio.net Thu Jan 11 22:00:00 1996 Path: biosci!daresbury!nntp-trd.UNINETT.no!nac.no!ifi.uio.no!news.sics.se!newsfeed.sunet.se!news01.sunet.se!sunic!newsfeed.ACO.net!swidir.switch.ch!in2p3.fr!univ-lyon1.fr!jussieu.fr!unilim.fr!cict.fr!opium.ipbs.fr!ducommun From: ducommun@opium.ipbs.fr (ducommun bernard) Newsgroups: bionet.molbio.proteins Subject: PEST score Date: 12 Jan 1996 17:00:04 GMT Organization: Laboratoire de Pharmacologie et Toxicologie Fondamentales (LPTF/CNRS, Toulouse) Lines: 9 Message-ID: <4d642k$r3c@news.cict.fr> NNTP-Posting-Host: opium.ipbs.fr Can anyone tell me where I could run a PEST score calculation on my favourite protein. http address ? Thanks Bernard ducommun@IPBS.fr From owner-proteins@net.bio.net Fri Jan 12 22:00:00 1996 Path: biosci!internet!biosci!not-for-mail From: biohelp (BIOSCI Administrator) Newsgroups: bionet.molbio.proteins Subject: BIOSCI miniFAQ, ver. 14-DEC-95 Date: 13 Jan 1996 02:00:21 -0800 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 199 Sender: daemon@net.bio.net Distribution: world Message-ID: <199601131000.CAA00195@net.bio.net> NNTP-Posting-Host: net.bio.net (LAST REVISION: 14-DEC-95) This BIOSCI "miniFAQ" is designed to answer the questions that come up the *most frequently*. The main BIOSCI FAQ (Frequently Asked Questions) is accessible on the World Wide Web at URL http://www.bio.net/. Contents: -------- 1) Using the WWW to access the BIOSCI/bionet newsgroups. 2) What to do about "spams," i.e., junk mail, ads, etc. 3) Examples of subscribing and unsubscribing to the mailing lists. 4) The BIOSCI user address and research interest directory. 1) Using the WWW to access the BIOSCI/bionet newsgroups. -------------------------------------------------------- As of 10 December 1995, all BIOSCI/bionet full newsgroups are accessible through the World Wide Web (WWW) at URL http://www.bio.net. One can read and reply publicly or privately to both recent postings and archived messages through one's Web browser if it is configured properly to send e-mail. Each newsgroup is equipped with its own WAIS index in addition to the master index for the entire set. The main BIOSCI home page also has access to the BIO-JOURNALS Table of Contents database WAIS index and the BIOSCI user address database described in another item further below. 2) What to do about "spams," i.e., junk mail, ads, etc. ------------------------------------------------------- BIOSCI is a set of parallel USENET newsgroups (the "bionet" groups), mailing lists, and a hypermail archive at URL http://www.bio.net/. The same postings are distributed on all media (except for a small number of mailing-list-only groups at net.bio.net). Unfortunately it is becoming a despicable practice on the Internet (by a few people out to make a fast buck) to do automated mass postings to thousands of newsgroups and mailing lists. These attempts to grab free advertising are refered to as "spams" in the usual, somewhat boneheaded, net terminology. USENET is more susceptible to this practice, and many spams originate on the USENET groups and then are passed on to the mailing lists. However, spammers also get lists of mailing addresses and hit these too, so neither medium is immune. What should you do personally if you get junk mail? --------------------------------------------------- Just delete it and move on without reading it further. Filing a protest is becoming increasingly useless because spammers are often disguising the addresses where the messages are sent from. Unless you really understand Internet mail systems, your attempt at protest by sending replies to the message will often end up being sent to the address of an innocent person that the spammer is victimizing. What can BIOSCI/bionet do to protect its newsgroups? ---------------------------------------------------- The only solution currently available is to moderate the newsgroup. If this newsgroup is already moderated, then you are in good shape. Moderation protects the USENET distribution from about 95% of the spams that are being sent to date and protects the mailing lists completely. Moderation means, however, that someone has to take the time to review each message before it goes out. We have set up software here that simply allows the moderator to forward to an address at net.bio.net messages that (s)he wishes to have distributed. This takes no more time than that needed to read the message and pass it on, say about 1 min. per message. Most newsgroups currently have a discussion leader who is responsible for their newsgroup. The discussions leaders and their e-mail addresses are listed in the BIOSCI Information Sheet which is available on the Web at http://www.bio.net/. If a newsgroup is being hit with too many junk postings, please contact the discussion leader for that group and see if there is interest in moderating the group. Please do not assume that by simply posting a complaint to the newsgroup itself, anyone on the BIOSCI staff will act on your complaint. With close to 100 newsgroups to run, the BIOSCI staff has to rely on the discussion leaders of each newsgroup to report problems directly to us at biosci-help@net.bio.net. We will moderate any of our newsgroups if the discussion leader tells us that the readership of the group wishes to do so and if a moderator is willing to do the work. For most BIOSCI/bionet groups, this entails only a few minutes of work each day. Moderating a newsgroup will resolve probably 95% of the junk postings on the USENET distribution. Unfortunately there are easy ways for determined spammers to override the moderation mechanism on USENET, but we can protect our e-mail subscribers from unwanted postings if the newsgroup is moderated. You can also access our newsgroups over the WWW at URL http://www.bio.net. While this Web interface will not stop spammers from trying to post to the groups, this will give you yet another way, besides using USENET news, to keep the junk out of your personal mail files. For those of you with local USENET news systems, the Web interface will also give you faster access to new newsgroups and recent postings. 3) Examples of subscribing and unsubscribing to the mailing lists. ------------------------------------------------------------------ PLEASE NOTE: The BIOSCI management does NOT act on subscription/unsubscription requests that are posted improperly to the newsgroups and mailing lists. People who do this only bother everyone on the lists to no avail. Please be sure to follow the proper procedures below. Gory details are in the BIOSCI Information sheets on the Web at http://www.bio.net. Below we give an example utilizing the METHODS-AND-REAGENTS list at both of our two BIOSCI sites: Users in the Americas and Pacific Rim countrie