pET vector cloning of hydrophobic protein gene

Mike Holloway mike.holloway at stjude.org
Tue Aug 1 14:01:38 EST 1995


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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Mike Holloway		    |* On average, 8 people a day on the waiting list
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* Only a small fraction of the next of kin of potential donors allow 
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* In general, medical professionals do not recognize their obligation to 
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