We have been expressing a family of human proteins as inclusion bodies in E. coli, which we can be refolded and crystallized. However, three members show no expression either as inclusion bodies or as soluble proteins.
The genes were ligated into the pE21d vector with either a His-tag or no His-tag. The genes were codon optimized for E coli. They are expressed in BL21(DE3), though I have tried the different strains for the non-expressing protein (e.g. Walker cells, Rosetta). What are the causes for no expression. I am guessing something to do with the mRNA. Should I try unoptimized/semi-optimized codons? Or is there something I could add to the media to aid in expression of the protein into inclusion bodies? Any suggestions?
