I have used the His-tagging vector pQE (Qiagen) for the overexpression
and purification of several recombinant mycobacterial proteins. In most
cases, the proteins were insoluble.
I then made my own His-tagging vector for expression in mycobacteria,
and have now found that 4 out of 4 proteins are soluble in this system.
The level of expresion is still fairly low (say 10-20 x more than from
the WT gene), but at least I have functional protein.
There are two explanations. Either these proteins prefer being expressed
in mycobacteria, or the overexpression makes them insoluble in E.coli.
To test this, I could try and put the genes in E.coli with a promotor
that provides low expression. Does anybody know if there are such
vectors commercially available? Ideally, I would want to use one with a
Dr Koen A.L. De Smet
Department of Medical Microbiology
Imperial College Medical School at St Mary's
London W2 1PG
Tel: (+44)-(0)171-594 3946
Email: k.desmet at sm.ic.ac.ukhttp://www.sm.ic.ac.uk/medmicro/home.htm
"If we knew what it was we were doing,
it would not be called research, would it?". -- A.Einstein