Expression and -N end rule

[shifali chatrath]

Dear all!
Thanks for the suggestions. SUMO was good one. Will try and get back to you after getting it done.

Thanks again

Shifai

On Thu, 23 Apr 2009 02:02:09 +0530  wrote
>Hi Shifali,
>
>Your protein is stable in the snake venom, because the venom itself
>might not have the 26S proteasomal activity. I think venom proteins are
>secreted using secretion granules which shield them from the cellular
>26S proteasomal activity.
>
>Regarding your troubles with expression, you could try these options.
>
>1- Codon optimization. For a small protein such as yours the entire gene
>could be synthetically made with codon optimization for
>E.coli/Yeast/Insect with affordable price (at least here in the US).
>
>2-SUMO expression system- Look into the SUMO expression system. 
>
>http://lifesensors.com/www/sumo-expression-systems-c-55.html
>
>In this system, SUMO (Small Ubiquitin-like Modifier) tag is added to the
>N-terminus of your protein. This will prevent N-end rule degradation if
>there is any. Once the protein is expressed you can purify it using
>standard Ni-Affinity Purification (There is a His tag N-terminus to
>SUMO). SUMO tag is then cleaved with a SUMO specific protease which will
>leave the original N-terminus of your protein intact (Arginine in your
>case). 
>
>This system offers vectors for E.coli, Yeast and Insect Cells.
>
>3- Once codon optimized, you can try several standard conditions to
>optimize the expression.
>
>-Medium
>-IPTG (Low to High)
>-Temperature (18oC to 37oC)
>-Time
>-Lysis buffers
>-Protease/Proteasome inhibitors
>
>Hope this helps and best of luck!
>
>Suresh
>
>Disclaimer: I do not work for Life Sensors Inc., but my company has
>close ties with them and I have used the SUMO expression system
>successfully in E.coli. 
>
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>-----Original Message-----
>From: methods-bounces from oat.bio.indiana.edu
>[mailto:methods-bounces from oat.bio.indiana.edu] On Behalf Of
>methods-request from oat.bio.indiana.edu
>Sent: Wednesday, April 22, 2009 1:05 PM
>To: methods from magpie.bio.indiana.edu
>Subject: Methods Digest, Vol 47, Issue 16
>
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>Today's Topics:
>
>   1. phenol in alcohol precip (Ed Siefker)
>   2. Blue/white with pET15b, Rosetta2(DE3)? (oliver.starks from gmail.com)
>   3. Genomic DNA isolation from mammalian cells (Dwayne Taliaferro)
>   4. Re: Blue/white with pET15b, Rosetta2(DE3)? (DK)
>   5. Re: phenol in alcohol precip (WS)
>   6. Re: Genomic DNA isolation from mammalian cells (DK)
>   7. Expression and -N end rule (shifali  chatrath)
>   8. Re: Expression and -N end rule (WS)
>   9. Genomic DNA isolation from mammalian cells (clarification)
>      (Dwayne Taliaferro)
>
>
>----------------------------------------------------------------------
>
>Message: 1
>Date: Tue, 21 Apr 2009 08:38:04 -0500
>From: Ed Siefker 
>Subject: phenol in alcohol precip
>To: methods from magpie.bio.indiana.edu
>Message-ID: 
>Content-Type: text/plain; charset=ISO-8859-1; format=flowed
>
>Hi.  I was a little hasty in doing some phenol/chloroform
>preps of genomic DNA and apparently didn't remove all
>the phenol before adding ethanol to precipitate.  How can
>I recover from this?
>
>I considered extracting with chloroform again, but I don't
>know if the phases will still separate with ethanol in there.
>Also, the DNA isn't in solution, so I'm guessing it would
>collect at the interface.  If there were more DNA there,
>I'd just fish it out with a pipette tip, but it's a small prep.
>
>Any thoughts?  Thanks a bunch
>-Ed
>
>
>
>------------------------------
>
>Message: 2
>Date: Tue, 21 Apr 2009 07:26:50 -0700 (PDT)
>From: oliver.starks from gmail.com
>Subject: Blue/white with pET15b, Rosetta2(DE3)?
>To: methods from net.bio.net
>Message-ID:
>   
>
>Content-Type: text/plain; charset=ISO-8859-1
>
>I've got my gene inserted in pET15b, and I transformed Rosetta2(DE3).
>Colonies appeared in appropriate numbers on an Amp/Cam plate,
>indicating the cells must have the vector.  Without thinking it out I
>re-streaked a couple colonies onto another plate with IPTG/Xgal to re-
>test insert presence (I'm fairly confident in my original ligation but
>it's always possible that the cells from which I prepped my plasmid
>were cotransformed with empty pET15b as well).  These produced blue
>coloration.  But after thinking about it more, it seems like either
>blue should never be produced (regardless of insert presence/absence)
>or should always be produced (regardless of insert presence/absence) -
>because the insert is not disrupting a lacZ gene like it is in
>pBluescript or something of that nature.
>
>So...how's the blue being produced, and is it even evidence of my
>insert not being present?
>
>I'll be testing these with new preps and PCR or digest today or
>tomorrow anyway but thanks in advance for your help.
>
>
>------------------------------
>
>Message: 3
>Date: Tue, 21 Apr 2009 13:20:43 -0400
>From: Dwayne Taliaferro 
>Subject: Genomic DNA isolation from mammalian cells
>To: 
>Message-ID: 
>Content-Type: text/plain;   charset="US-ASCII"
>
>Does anyone have a quick and reliable protocol for isolating gDNA from
>mammalian cells?  I  would be interested.
>
>
>
>------------------------------
>
>Message: 4
>Date: Tue, 21 Apr 2009 22:59:52 GMT
>From: dk from no.email.thankstospam.net (DK)
>Subject: Re: Blue/white with pET15b, Rosetta2(DE3)?
>To: methods from net.bio.net
>Message-ID: 
>
>In article
>,
>oliver.starks from gmail.com wrote:
>>I've got my gene inserted in pET15b, and I transformed Rosetta2(DE3).
>>Colonies appeared in appropriate numbers on an Amp/Cam plate,
>>indicating the cells must have the vector.  Without thinking it out I
>>re-streaked a couple colonies onto another plate with IPTG/Xgal to re-
>>test insert presence (I'm fairly confident in my original ligation but
>>it's always possible that the cells from which I prepped my plasmid
>>were cotransformed with empty pET15b as well).  These produced blue
>>coloration.  But after thinking about it more, it seems like either
>>blue should never be produced (regardless of insert presence/absence)
>>or should always be produced (regardless of insert presence/absence) -
>>because the insert is not disrupting a lacZ gene like it is in
>>pBluescript or something of that nature.
>>
>>So...how's the blue being produced, and is it even evidence of my
>>insert not being present?
>
>Rosetta(DE3) is simply BL21(DE3)  with pRARE2 plasmid. 
>The strain has WT lacZ, so naturally it makes blue colonies. 
>It has nothing to do with your plasmid or it having an insert. 
>
>DK
>
>
>------------------------------
>
>Message: 5
>Date: Tue, 21 Apr 2009 14:09:10 -0700 (PDT)
>From: WS 
>Subject: Re: phenol in alcohol precip
>To: methods from net.bio.net
>Message-ID:
>   
>
>Content-Type: text/plain; charset=ISO-8859-1
>
>Dear Ed,
>
>don't worry. Just rinse your pellet a few times with EtOH (and spin
>down shortly to make sure you don't loose the pellet).. If you plan
>downstream processing with enzymes, make sure all phenol has been
>removed. You might dare to check thesmell; your nose should turn out
>to be a very sensitive GC system with a neuronal detector attached to.
>Chloroform might not be that good choice, even that DNA doesn't
>dissolve in it, (regardless if there is EtOH and or phenol present),
>as it does not dissolve phenol as well as ethanol does. You might
>remember: the DNA (and other water soluble stuff) is in the phenol
>phase, lipids are in the chloroform phase and proteins are inbetween.
>
>Have fun!
>
>Wo
>
>
>------------------------------
>
>Message: 6
>Date: Tue, 21 Apr 2009 23:22:51 GMT
>From: dk from no.email.thankstospam.net (DK)
>Subject: Re: Genomic DNA isolation from mammalian cells
>To: methods from net.bio.net
>Message-ID: 
>
>In article , Dwayne
>Taliaferro  wrote:
>>Does anyone have a quick and reliable protocol for isolating gDNA from
>>mammalian cells?  I  would be interested.
>
>The purpose of gDNA? For "high" yeild and reasonable purity,
>here is what I used on many different cells (dicty, insect, 
>mammalian lines):
>
>Pellet cells from 10 ml culture, resuspend in 1% Triton X100, 
>spin, keep pellet, lyse nuclei with 400 ul 1% SDS, 20 mM EDTA,
>add 600 ul chloroform, vortex 15", spin hard, take upper phase, repeat 
>extraction 2X. Add RNAse to 0.1 mg/ml, let sit for 5', mix 3 volumes 
>of Qiagen buffer GQ or 5 volumes of Qiagen buffer PB with one 
>volume of chloroform-extracted material, load onto regular Qiagen
>miniprep column, wash 2X with PB, wash 2X with PE, elute with 
>100 ul of 1-10 mM Tris, pH 8.0-8.5 (or TE) heated to ~ 70C. 
>
>DK
>
>
>------------------------------
>
>Message: 7
>Date: 22 Apr 2009 02:45:33 -0000
>From: "shifali  chatrath" 
>Subject: Expression and -N end rule
>To: 
>Message-ID: 
>Content-Type: text/plain; charset="ISO-8859-1"
>
>Dear all!
>I had mentioned my problem earlier also in the forum but nobody replied.
>I am sure you great minds should have a solution to my problem. Please
>help me if you can!
>
>I want to express a snake venom protein,~ 10kDa, 10 cys residues and 5
>disulphide bonds.Mature protein has Arg as first residue after signal
>sequnece is clipped off.
>First of all, i tried yeast expression system.
>I expressed my protein in pPICZ alpha, under alpha factor signal
>sequence followed by Kex2 and Ste13 signal sequences, such that the
>mature protein will have native -N terminus with Arg as first residue,
>as of my protein. BUt, I could not see any protein expression even under
>different medium and proper aeration and whatever possible
>troubleshooting one could do.
>Recently, i read -N end rule which states that Arg is destabilizing
>residues and directs the protein for degradation to 26S proteasome.
>I checked with the company, they say, It hold true for every expression
>system.
>But, my question is, if that is the case, how the protein is stable in
>snake's venom. I have found the protein in the crude venom of the snake.
>Also, the protein is expressed under alpha factor signal sequence which
>is supposed to be secreted into the medium. Therefore,once the protein
>is out into medium, it is out of cell now, Kex2 and Ste13 cleavages
>should be happening outside the cell, therefore, there should be no
>proteasomal degradation. Am I right?
>
>After clipping off signal sequence, protein will be exported out, still
>will there be any proteasomal targetting of proteins with destabilizing
>residue at -N terminus?
>
>I read in -N end rule that, protein's life depends upon penultimate
>residue to Met. We know, for a protein to be synthesised, we need to
>have  a start codon,i.e. Met. In -N end rule they say, Met will be
>removed be Metaminopeptidase(MAP), so if we express a protein we should
>not be counting Met residue in the expected MW? Because, not all mature
>protein sequences start from Met.
>
>
>Then,I tried expressing my protein using E. coli epression system, cDNA
>cloned in pET19b in Rosetta gami cells and RIL cells.I couldn't see any
>expression. Cells were growing normaly.(I hope, the protein is not
>inhibiting transcription and translation). Met and Gly had to be added
>at -N terminus to maintain the reading frame.
>Protein is 10Kda and was cloned without tag sequence. I mean expected
>protein should be 10kDa.I confirmed the sequences before cloning and
>even plasmid isolated from ex-pression hosts. DNA is there but no
>protein.
>
>Please answer any, if not all, of the above question. i am struggling
>with all this for alst 8 months. Your suggestions and help will be
>highly appreciable.
>
>Thanks in advance.
>
>Shifali
>
>
>
>Shifali Chatrath
>Graduate Student
>Protein science Lab
>Dept. of Biological sciences
>National University of Singapore
>Singapore
>+65-65161210
>+65-96393449
>
>------------------------------
>
>Message: 8
>Date: Wed, 22 Apr 2009 01:13:52 -0700 (PDT)
>From: WS 
>Subject: Re: Expression and -N end rule
>To: methods from net.bio.net
>Message-ID:
>   
>
>Content-Type: text/plain; charset=ISO-8859-1
>
>Hi Shifali, maybe your protein is toxic? you could change codon usage
>and/or try higher eucaryotic cells.
>
>To narrow down the effect of signal sequences etc.: Have you
>considered putting your protein (i.e its cDNA) and/or part of it in
>front of a reporter gene like GFP or insert just anything known in
>order to disrupt a possile action?
>
>Best regards,
>
>Wo
>
>
>------------------------------
>
>Message: 9
>Date: Wed, 22 Apr 2009 11:03:41 -0400
>From: Dwayne Taliaferro 
>Subject: Genomic DNA isolation from mammalian cells (clarification)
>To: 
>Message-ID: 
>Content-Type: text/plain;   charset="US-ASCII"
>
>I am looking for a protocol that will allow me to purify genomic DNA
>from
>mammalian cells with the purpose of using it for Southern blotting and
>PCR.
>
>DT
>
>
>
>------------------------------
>
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Shifali Chatrath
Graduate Student
Protein science Lab
Dept. of Biological sciences
National University of Singapore
Singapore
+65-65161210
+65-96393449