GST and proteases

Bernard Heymann bheymann at bragg.bio.purdue.edu
Tue Sep 27 00:07:39 EST 1994


I have succeeded in expressing my membrane protein behind GST, producing a
soluble protein in reasonable quantities. However, I get two proteins from
the GSH-agarose gel, the fusion protein and the GST portion alone. After
trying to speed up the purification and using protease inhibitors, the
amount of fusion protein increased, but still about half of the purified
protein is GST. Is there anybody out there that can suggest one or more
cell-strains lacking proteases that I can try. The strains I've been using
are JM103 and BL21. I actually switched to BL21 because it lacks OmpT,
which has been shown to cleave GST fusion proteins. Since my GST fusion
protein is expressed in the cytoplasm, with the membrane protein part of it
perhaps inserted in the membrane, I expect the culprit protease to reside
in the cytoplasm or cell membrane. 

-- 
Bernard Heymann
bheymann at bragg.bio.purdue.edu



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