Hi In addition to what Fred and others have said, it is important to remember there are several native, stress-responsive proteins of E. coli that show good affinity for metal ions. Hence, they can be easily co-purified by immobilised metal affinity chromatography (IMAC). This seems particularly critical when the recombinant protein/protein domain of interest is expressed at very low levels and/or shows low metal binding capacity. A number of reasons for this include having the histidine tag partially buried, low intrinsic stability of your protein; its tendency to aggregate; etc.).
We wrote a short review on this issue not long ago hoping it would help to identify quickly the usual suspects (doi:10.1016/j.bbagen.2006.03.027). I totally endorse what others have said: by all means always check the DNA sequence of what you want to express, and confirm its identity once you have purified it (N-terminal sequence, mass spec., etc.) to avoid disappointment and save resources. Kind regards Victor (Tom Blundell lab). Department of Biochemistry University of Cambridge 80 Tennis Court Road CB2 1GA Cambridge -----Original Message----- From: CCP4 bulletin board [mailto:[email protected]] On Behalf Of Kerff Frédéric Sent: 18 February 2010 23:16 To: [email protected] Subject: Re: [ccp4bb] Expression of a protein of 43KDa The maltose binding protein is one possibility. Some collaborators did provide us with very pure MBP instead of the protein of interest... Mass spec eventually identified it after we wasted some time on it... Fred > I am trying to overexpress a His-tagged protein of 29KDa in E.coli > (BL21-codon plus) and I end up with a highly expressed product that of > 43KDa that binds to the Ni-column. I also have nice crystals. Does > anyone have any experience on this. > Armando >
