Camilla Rang wrote:
>> Hi, I constructed plasmid expressing GST-foreign peptide using pGEX-KT
> vector, but can not get the induction of fusion protein, only the GST be
> induced. I hope someone can give me helpful suggestion.
Have you sequenced the plasmid to check that the fusion protein is in
frame with the GST and no mutations have crept in?
I speak from bitter experience here - I once spent weeks fiddling around
with a GST expression system only to finally discover that my boss had
made a single base mistake when ordering the PCR primers and thus
inserted a stop codon a few amino acids into the fusion gene!
If the sequence is correct, it's possible that the fusion protein is
degrading. What methods have you used to check for the expressed gene?
If you have access to a suitable antibody, you could try looking at the
whole pellet lysate on a western blot and see if there is any sign of
your fusion protein.
If the protein is degrading, you can try inducing the bugs at room
temeprature instead of 37 degrees, inducing for a shorter time and/or
adding protease inhibitors during the processing of the protein.
> Best regards.
> Yong Jiang