fusion protein expression vectors

GIETZ at bldghsc.lan1.umanitoba.ca GIETZ at bldghsc.lan1.umanitoba.ca
Thu Jan 21 17:14:00 EST 1993


%%%%%%%%%%%%%%%%(Brian J.Baumgartner) writes:%%%%%%%%%%%

>We would like to create fusion proteins for synthesis of antibodies.  We
>must be able to cleave our desired peptide from the fusion product and
>affinity purify the fusion.  Several companies make these types of
>prokaryotic expression vectors.  We would appreciate any comments on which
>vector has proven the most effective for you.  No sales reps please!
>
>Thanks, 
>
>Brian Baumgartner
>Dept. of Biochemistry
>Baylor College of Medicine
>Houston, Tx. 77030
>e-mail: brianb at mbcr.bcm.tmc.edu
%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%

Hi Brian!
    We have used pGEX-2T system, which fuses your gene
to the glutathione S transferase protein and has a thrombin cut
site between this protein and yours, with great success.  See
Smith and Johnson (1988) Gene 67,31-40.  The proteins can be
affinity purified using glutathione agarose in a single step to 
a very high level of purity.  Our fusion protein can be cut directly 
'from the beads with a very high efficiency.  The paper by Oettinger
et al (1992) Biotechniques 12;p544, describes using this system for
the production of antisera.  In addition we have found that the growth
conditions are important for the production of maximum amount of
fusion proteins.  Our best are 25oC growth with2 hr induction with IPTG in 
strain 
JM109.  We have also found that specific growth media also helps increase
the yield of active (properly folded?) protein.  Blackwell and Horgan (1991) 
Febs letts 295;
p10-12.(Thanks to Kiran Madura) describe a method using betaine in LB media
 with sorbitol to increase 
the yield of active( properly folded) fusion proteins.  We have tried it and 
it
works very well for  both our fusion proteins.  pGEX is marketed by 
Pharmacia/LKB.

Hope this helps!

Dan Gietz
U of Manitoba
GIETZ at BLDGHSC.LAN1.UMANITOBA.CA



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