> I have sequenced gene - RBS included- to be sure, that there is not frame
shift or any other mutations. I have not sequenced >promoter region - I can
do that.
> Lately I have heard about adding protease inhibitor PMSF directly into
medium. Does it make any sense?
Well... PMSF isn't very soluble. It will go into solution by sticking to
greasy bits of whatever's floating around (read, bacterial membranes) and
might or might not result in improvement. I think that His-tagging your
protein, or better yet, sticking an MBP before it (e.g. pMAL system, but
make sure that your cleavable linker is a) short b) something specific like
TEV) will give you at worst some idea of what's going on, or at best actual
protein to purify. Because face it - you probably just want the protein :) ?
A.G.E.
