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 * |
