Protein Expression Problems

William P. Tschantz tschantz at galactose.mc.duke.edu
Sat May 2 08:04:38 EST 1998


HI

I have a difficult protein expression problem to solve and thought 
someone out there may have some tips.

I have recently cloned a novel gene from a human brain library.  I know 
that i have the whole coding sequence since i can transcribe and translate 
it in vitro using a TNT system and it comigrates with the native purified 
protein (bovine).  The original clone contained a large 3' UTR about 1.5 
times larger than my coding sequence and a very short 5' region.  I have 
made 7 different constructs for expression (2 differt CMV promoter 
vectors in COS cells, baculavirus, and E coli pET system)  One set of 
constructs is the full length clone with 3' UTR and the other sets is 
where i have removed the 3' UTR.   None of these constructs express at 
all based on western blot analysis with a decent antibody and by activity.

Has anyone seen this before.  I am planning to try other promoters in the 
mammalian system (SV40, adenovirus).  Any hints would be greatly 
appreciated!!

Thanks in advance.

Bill
-- 
| William R. Tschantz, Ph.D.                 *                        |
| Dept of Pharmacology and  Cancer Biology   *                        |
| Duke University, Durham, NC                * Got Homebrew ??        |
| tschantz at galactose.mc.duke.edu             *                        |



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