1. look up Bacillus megaterium expression system (commercial, pretty cheap & relatively easy). It's easier to use than some of the integration-based B. subtilis systems. Or you could ask around for a suitable B.sut. shuttle vector as an alternative.
2. Bacillus codon set is heavily biased towards AT-richness and even in the codon+ may not be expressed well - there may also be mRNA issues (stability). It's not hard to make synthetic genes these days - if you're desperate to continue using E. coli for this work you can make a synthetic codon-optimized version specific to the host. Cheers, Artem On Wed, Apr 14, 2010 at 2:30 PM, Amit Kumar <amit23...@gmail.com> wrote: > Hi all, > > Sorry for a non-ccp4 query. > > I will highly appreciate your comments on the expression: > > I am trying to express HIS tagged ~60 kDa cytosolic protein from Bacillus in > E.coli pET expression system. I tried codon+ strain, time of expression > and various temperatures ( 18, 25, 30, 37C) for the expression. Neither > full-length nor truncated form of the protein was found to be expressing > (also checked by western blot). What other parameters should I explore to > get the expression in Escherichia coli? > > Thank you very much for your comments. > > Amit K. >
