Overexpressing a protein
JM at 843724082793.unil.ch
Sun Nov 28 07:15:08 EST 1999
In article <383D52A4.FCCFA0E4 at agora.ulaval.ca>, aca611 at agora.ulaval.ca wrote:
> I am looking for a good method for the overexpression and purification
> of a 50 amino acid protein. High yield (at least 10 mg) and good purity
> are required. No post-translational modification needed. Is a GST fusion
> protein in E.coli a good choice?
> Serge Champetier, M.Sc.
> Quebec City, Canada.
I've done it with a 6xHis tagged 62 aa protein in E. coli under the LacI
controlled (IPTG) tac promoter. Be sure to have a good RBS before your
start codon (I've seen a paper where they forgot it!) and to use
alternatively CAC and CAT codons to make your tag (E. coli doesn't seem to
like having 6 consecutive identical codons). By using the QIAexpressionist
kit (Qiagen) I've been able to routinely purify 0.4-0.5 mg (Bradford BSA
eq.) per 200 ml culture under native conditions. Denaturing conditions
should give you much higher yields, or you could scale up the protocol to
fit your needs...
More information about the Methods