Just to add couple of things to what Artem said.. If it is something similar to a mammalian kinase or malaria protein for example,
1. Recodonizaton can change expression from near zero to substantial amounts, however, a) ideally, it needs to be recodonized separately for each target expression system (E. coli / yeast / IC) b) Expression does not guaranty solubility. One thing which sometimes works is coexpression of a phosphatase, if the non phospho form is of any interest. Similar things have been done by Src related kinases.. YopH or Lambda might be a good starting point. See: http://www.ncbi.nlm.nih.gov/pubmed/16260764?ordinalpos=3&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_DefaultReportPanel.Pubmed_RVDocSum HTH, Partha On Tue, Feb 24, 2009 at 3:48 PM, Mo Wong <mowon...@gmail.com> wrote: > I thought I'd post this to the CCP4bb, as judging by previous posts, it > seems I could get some useful insight into my problem... > > This is question has probably been asked by people for a long as molecular > biology has been around, but hopefully my question isn't a complete rehash > of other peoples: I am trying to express a human protein in bacteria where > the only modified amino acids are 3 phosphorylated serines. I’ve gone > through the usual hoopla of trying to get it expressed in E. coli > (Rosetta/Codon+ cells, varying IPTG, low temperature, etc). Sequencing > confirms my insert is correct, but from coomassie gel inspection, I appear > to get near zero induction (I need to do a Western to get a clearer > assessment). I’ve heard about custom gene synthesis, and it appears Mr. Gene > (https://www.mrgene.com/) would be a good avenue to look into as they > optimize the ORF taking into account codon usage in E. coli (though I’m not > sure they examine putative mRNA substructure formation like some companies > do). It’s only 49c per base pair, so doesn’t seem too cost prohibitive. My > only concern is that if this protein is toxic, I could be wasting money. > > So I was wondering, has anyone seen the expression for a particular > protein change from zero in Rosetta/Codon+ cells using "native" sequeneces > to being largely overexpressed in BL21(DE3) cells using codon optimized > sequences? For folks who have had a similar problem to the one I've > described, would you recommend that I first try using a codon optimized > sequence in E. coli over testing protein expression in yeast/insect cells, > or the other way round? > > Thanks! > -- MRC National Institute for Medical Research Division of Molecular Structure The Ridgeway, NW7 1AA, UK Email: pcha...@nimr.mrc.ac.uk Phone: + 44 208 816 2515
