GST fusion prots truncating at 27 kDa?
lghays at U.WASHINGTON.EDU
Thu Jul 13 10:56:53 EST 1995
On 13 Jul 1995, Jeff Pitman wrote:
> I have had repeated problems trying to express full-length GST
> fusion proteins, when fusing small (60 amino acid, homeodomain)
> protein motifs with GST.
> Using two different homeodomains (but going into the same site in
> the pGEX-KG vector), I have gotten the same results: when I express
> the protein, I only get an ~27 kDa protein (the same size as the
> default GST protein).
> I have sequenced the vectors, and they are indeed in frame and
> unmutated (both inserts were PCR products), so it's not a stop codon
> problem. I have tried altering induction time (1-7 hrs), induction
> temperature (RT, 37C), bacterial hosts (DH5alpha, XL2Blue, XL1Blue),
> all to no avail (same 27 kDa protein). Meanwhile, I have gotten
> beautiful full-length production of other constructs using
> full-length and truncated versions of the same homeodomain proteins
> (with the same pGEX vector, albeit going into different sites).
> Has anyone else seen this before, and better yet, figured out a way
> around it?
> -Jeff Pitman
> Frustrated grad student
I can understand your frustration. I have had the same problem
expressing a large protein from a pET vector. I had no success in
solving the problem though. It was suggested to me by Novagen that
there may be some mRNA secondary structure interfering. The only
suggestion I can give is to clone into a different site. Good Luck
Lara G. Hays
lghays at u.washington.edu
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