GST fusion prots truncating at 27 kDa?

Jeff Pitman jpitman at sdcc3.ucsd.edu
Wed Jul 12 21:58:03 EST 1995


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?

Thanks, 

-Jeff Pitman
 Frustrated grad student





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