baculo expressed protein insoluble

albrecht at hpi.uni-hamburg.de albrecht at hpi.uni-hamburg.de
Fri Mar 26 05:16:31 EST 1999


Dear reader,

I have a serious problem to express a set of His-tagged deletion mutants of
my protein in the baculo-system. All fragments are insoluble as judged by
comparison of native and denaturing protein purification methods. Under
denaturing conditions (6M urea)I see a huge expression of the proteins but
there is no binding to the metal-affinity substrate under native conditions.
Interestingly I can see little round "inclusion bodys" (?) in the infected
insect cell culture (is that possible ?). To tackle this problem, I did an
infection time course to figure out whether a portion of the protein is
soluble in early stages of infection. Unfortunately it was not. Since I use
Invitrogens Hi5 insect cell line with is supposed to express 5 to 10 fold
more protein I want to test a "normal" SF9/X/21 cell line. Also the decrease
of culture temperature to room temp. might slow down the protein synthesis an
lead to soluble protein. Since I want to check a enzymatic activity,
refolding or renaturing is not the method of choice. Does anyone has
experience with that kind of problem ? I appreciate any suggestions

Thanx in advance
Nils

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