pET expression system

Steve Minchin S.D.Minchin at bham.ac.uk
Thu Mar 2 10:37:03 EST 1995


In article <3ijklg$900 at forged.passport.ca> scontz at passport.ca (Marc Visconti) writes:
>Path:
>bham!warwick!lyra.csx.cam.ac.uk!sunsite.doc.ic.ac.uk!hgmp.mrc.ac.uk!daresbury!bi
>oftp.unibas.ch!citi2.fr!jussieu.fr!oleane!pipex!swrinde!howland.reston.ans.net!n
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>From: scontz at passport.ca (Marc Visconti)
>Newsgroups: bionet.molbio.methds-reagnts
>Subject: pET expression system
>Date: 23 Feb 1995 22:43:44 -0500
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>I'd like to thank everyone for being so kind as to send me their 2 cents
>worth regarding my problem.  However, I realize I may not have fully
>explained my problem.  The term "increadible induction" was in reference to
>my Western blots.  With the other expression systems used, I never saw a
>clear cut difference between my induced and non-induced fractions.  I made
>a conclusion that my protein is soluble by running soluble and insoluble
>lysates after sonication.  Virtually all of my product, when detected with
>the S-protein Western kit, was soluble.   
> 
>Currently my protocol requires plating transformed cells on kanamycin
>plates (50 ug/ml), picking a colony, growing o/n to an OD-600 of 1.5- 2.0. 
>This is used as innoculate into fresh media (LB for now) with 50 ug/ml
>kanamycin.  These are grown to OD-600 0.4-0.6, and then induced with 1mM
>IPTG for 2-3 hours.  These are then spun down, and resuspend in Sample
>running buffer with SDS, boiled and run on SDS-PAGE.  Am I doing something
>wrong or missing something?  Thank you in advance for all of your help. 
> 
>--Marc Visconti 
>scontz at passport.ca 
>marcvis at resunix.ri.sickkids.on.ca

We had a similar problem when we tried to overexpress the human cardiac TnI in 
E. coli using the pET system. We first looked at codon preference with no 
success. So we made successive deletions from the N-terminal coding region, 
this showed us that lack of expression was due to the first five codons. We 
developed a screen for expression and then mutated the first five codons 
(maintaining the coding potential). We picked up mutant which over expressed 
TnI, this contained two changes, which were both in wobble bases and therefore 
the mutant gene produced the same peptide sequence as the wild-type gene.
If you want more information we have published the work.

Al-Hillawi, E., Minchin, S.D., and Trayer, I.P. 
Overexpression of human cardiac troponin-I and troponin-C in Escherichia coli 
and there purification and characterisation.
Eur.J.Biochem. 225:1195-1201, 1994.



All the best


Steve Minchin


---------------------------------------------------------
Dr. Steve Minchin,

School of Biochemistry, The University of Birmingham,
Edgbaston, Birmingham, B15 2TT, U.K.

E-Mail
InterNet:       S.D.Minchin at bham.ac.uk
Janet:          S.D.Minchin at uk.ac.bham

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