Many kinases phosphorylate themselves when expressed in E. coli. You probably get more phosphorylation in E. coli than you express the same protein in insect cells or mammalian cells.
However, if your protein is not a protein tyrosine kinase, you may check your western condition. My experience is that phosphorylations of over-expressed proteins in E. coli are always auto-phosphorylation. We have a publication in Protein Expr Purif. 2005 Dec; 44(2):121-9 that investigated the phosphorylation sites. Cheers, 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 [EMAIL PROTECTED] www.accelagen.com -----Original Message----- From: CCP4 bulletin board [mailto:[EMAIL PROTECTED] On Behalf Of Narayanan Ramasubbu Sent: Friday, October 26, 2007 3:31 PM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Is phophorylation possible in E. coli expression system? Dear All: This is not crystallography related and does not belong to this group, but I would like to pose this to all who are working with proteins expressed in an E. coli BL21 (DE3) or Rosetta. Is it possible for a protein to be phosphorylated during expression? At least my understanding was that post-translational modifications are not possible in E. coli. However, recently we expressed a protein and noticing that there are lot of potential tyrosine phosphorylation sites, we checked the expressed protein on a gel using a stain that detect phosphates. (Please do not say Are you crazy? Why would you check.. etc). Lo and behold, there lights up the band. Hence my question to you all. I could not google or mine from PubMed specific references that exist regarding this. Please enlighten me. Thanks a lot. Also, I would like to take this opportunity to thank everybody for all the help whenever ask some off-topic question like ths. Subbu
