Alfredo, The other alternative explanation to your two species protein sample is in the presence or absence of initiator methionine. About 70-80 of all prokaryotic proteins under post-translational removal of initiator formylmethioine (178 Da) in two step process. First the formyl (29 Da) group is removed by a deformylase and then the initiator methionine (149 Da) by methionine aminopeptidase. It is possible that one or two of these enzymes have partial activity resulting in two species. In the N-terminal sequencing it may or may not show up clearly.
Anthony On Mon, 19 Jan 2009 21:31:05 -0500, Artem Evdokimov wrote > Hi, > > Yes, this does happen. > Spontaneous รก-N-6-Phosphogluconoylation of a "His Tag" inEscherichia > coli:The Cause of Extra Mass of 258 or 178 Da in Fusion Proteins > > http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6W9V-45N4K22-R&_us > er=10&_rdoc=1&_fmt=&_orig=search&_sort=d&view=c&_acct=C000050221&_version=1& > _urlVersion=0&_userid=10&md5=453fd46805ef7137c62705a5ae80384e > > There are other options out there too but this one comes to mind first. > > Artem > > -----Original Message----- > From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of > Torres-Larios Alfredo > Sent: Monday, January 19, 2009 8:26 PM > To: CCP4BB@JISCMAIL.AC.UK > Subject: [ccp4bb] Posttranslational modifications of recombinant proteins > expressed in E. coli? > > Dear all, > > I know this sounds weird (and besides, it's a non CCP4 question), but > does anyone know any reports or has worked with a recombinant protein > that is apparently post translationally modified by E. coli? > > We have an enzyme with two populations that have been sequenced at the > N-terminus. Both have the same sequence at that region, but by mass > spec the MW of one population (1) is 200 Da more than expected. The > other population (2) has the "correct" MW. The enzyme (1) has 10% > activity with respect to (2). The C-terminal is far away from the > active site. > > Another funny thing is that the ratio of the two populations changes > from batch to batch, and the heterogeneity arises before cleaving the > N-terminal His tag with TEV protease. > > Any comments will be greatly acknowledged. Thanks a lot in advance, Alfredo. > > Alfredo Torres-Larios, PhD > Assistant Professor > Instituto de Fisiologia Celular, UNAM > Circuito Exterior S/N. Ciudad Universitaria > Mexico, DF, Mexico > > ---------------------------------------------------------------- > This message was sent using IMP, the Internet Messaging Program. ------------------------------------------------- Anthony Addlagatta, Ph.D. Ramanujan Fellow and Senior Scientist Center for Chemical Biology Indian Institute of Chemical Technology [IICT] Tarnaka, Hyderabad- 500007, INDIA Tel:+91-40-27191583 Url: http://www.iictindia.org/zacb/Dr.%20Anthony.aspx
