pET vector cloning of hydrophobic protein gene

ChunLin_Lu ChunLin_Lu at cellbio.duke.edu
Wed Aug 2 11:29:51 EST 1995


Yes, I do have similar experiments like you. I want to express Sul A ( a
bacteria division inhibitor) in BL21 using PET11b vector. I amplified sulA
gene from DH5a, and cloned into PET vector, transformed into DH5a. I got
recombinant vector (hope it not be rearranged), but can not see a
transformant from BL21 transformation. I repeated this seveal times, no
sucess at all. Many peoples here are using pET system to express prteins,
do not have any problems. I think some SulA may be made even without any
inducer, because excess SulA is toxic to bacteria, which inhibites cell
division. 
I am thinking about how to solve my problem, could give some suggestions.
Thanks. Chunlin
In article <3vltmi$qn5 at warp.cris.com>, mike.holloway at stjude.org (Mike
Holloway) wrote:

> I'm giving up on the pET system and wondered if anyone else has had 
> similar experiences.  Even with the thioredoxin fusion protein vector, 
> I can not isolate a transformant with my sequence and an intact vector. 
> I do get many rearrangements - things that are not whole pET vector or 
> PCR template from the preparation of my insert.  The transformants are of
> various sizes, with various restriction sites missing.  Other cloning 
> projects using the same bugs and reagents are working just fine, without
> any evidence of contamination. 
> 
> I have to conclude that some protein is being made, despite what anyone 
> says about the T7 promoter not being used without the polymerase being 
> pumped out, and that its toxic.  This has been the previous experience 
> in our lab with this protein using other bacterial expression vectors.  
> Apparently this isn't an isolated experience, since several companies 
> have recently come out with this thioredoxin fusion protein scheme that's 
> supposed to solve everything.  
> 
> Anyone had experience with pET vectors?
> 
> =========================================================================
>=============



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