Hi Chen,
Since you recognize that this is a protein expression problem, the best way to get return of your investment of efforts is to get soluble expression instead of trying to solubilize the protein down stream. Many good suggestions have been proposed. I just want to add one more thing for you to try. Lower the induction temperature to 16C (or any temperature between 10-30C). With all the tricks to make protein soluble in E. coli, low induction temperature is the only universal method that works. Accelagen has made all kinds of proteins and we have systematically tried many variables. Only induction temperature matters for solubility, everything else just gives you more or less proteins. If your promoter is not tightly controlled, you may just let the E. coli grow to saturation without induction, at low temperature of course. It worked well for pGEX based vectors. Many proteins give little soluble expression in E. coli. In those cases, Baculovirus expression system is a great alternative. Once you have your gene in a transfer vector, it takes about 3 weeks to know the soluble expression level in insect cells and another 3 weeks to harvest 10 L cell pellets. Currently technology allows you to scale up to 100s L in an additional couple weeks. The material cost is actually similar to E. coli expression. It's faster than trying to figure out how to solubilize a protein. Good luck. Chun Chun Luo, Ph.D. The Protein Expert Accelagen, Inc. 11585 Sorrento Valley Road, Suite 107 San Diego, CA 92121 TeL: 858-350-8085 ext 111 Fax: 858-350-8001 <mailto:[EMAIL PROTECTED]> [EMAIL PROTECTED] www.accelagen.com _____ From: CCP4 bulletin board [mailto:[EMAIL PROTECTED] On Behalf Of Daniel Jin Sent: Monday, January 21, 2008 10:56 PM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] protein expression problem Hi, I have been trying to express a rat protein in bacteria. The MBP-fusion expressed at very high level (~ 40 mg/L) while the GST-fusion and His-tag only gave inclusion bodies. The problem is that all protein runs in the void volume on a size-exclusion column (s-200, hepes, pH 7.4, 200 mM NaCl), no matter it is the intact MBP-fusion or cleaved sample. There is no Cys on this protein so there is unlikely any disulfide bond related problem. Anything I can do before I throw away this construct and try insect or mammalian cells? Thanks. Best, Chen _____ Never miss a thing. Make Yahoo <http://us.rd.yahoo.com/evt=51438/*http:/www.yahoo.com/r/hs> your homepage.
