The presence of a His-tag in the sequence does not automatically mean that the protein will bind to IMAC resin. Since your protein is so huge and since the His-MBP-fusion does not work any better than the His-fusion I would hazard that your protein is highly aggregated in solution, to the extent that it a) does not enter the beads and b) the His-tags are obscured by all the other junk.
One of the first things I'd do would be to try detergent lysis of your culture. This oftentimes can at least reduce the aggregate size down to the point where they enter the beads. Artem _____ From: CCP4 bulletin board [mailto:[EMAIL PROTECTED] On Behalf Of changrui lu Sent: Wednesday, October 10, 2007 9:13 PM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] His tag does not bind. Dear all, I am trying to express a 150 kd protein in E coli. I have it in two constructs, one with pmal-his and other with only his tag at N terminus. The full length protein can be detected both by sds and western using anti-his (190kd and 150kd respectively) but strangely neither binds to his-column very well. The majority of the full length comes through the column either at loading step or low salt wash step. The major species that gets trapped and eluted is the mbp-his truncation (~40kd). Some, though very little, full length protein did make it out the his column. The pmal-his construct does not bind amylose resin any better with majority flows right through. All purification are carried out under standard conditions as mentioned in the manuals. The protein is soluble and does not precipitate in the columns. I appreciate and ideas or explanations. Thanks in advance. Ray Cornell Univerisity
