Nonredundant summary - thx to all submitters: 1) disulfide was often present even after expression in a normal BL21 strain
2) try OrigamiB (DE3), Rosetta-Gami 2 from Novagen, are codon optimized and have mutations in the thiorodoxin reductase and glutathione reductase genes - expressing from pET32 vectors which contain a thioredoxin fusion tag - extra purification steps are generally needed to remove the thiorodoxin after cleavage of the fusion tag - level of expression achieved was not very impressive for E. coli, but enough to work with for structural studies - generally human, extracellular proteins - as always, protein dependent - Origami strains are somewhat slow growing and sick-seeming do not grow to very high densities and have to be coddled somewhat - They grow well enough in auto-induction media - grow to OD around 1.0-1.1 at 37, cool flasks in ice bath to 20, induce o/n with 30 uM IPTG at 18°C. - some success with periplasmic export for disulfide bridge formation using the OmpA leader sequence (not the PelB leader from the pET plasmids) 3) used the AD494 (trx) strain with good results 4) "Tuner" strain (also Novagen) and low levels of IPTG to induce expression - led to expression of a protein with one disulphide bridge 5) try pichia. br -----Original Message----- From: CCP4 bulletin board [mailto:[EMAIL PROTECTED] On Behalf Of Bernhard Rupp Sent: Wednesday, November 28, 2007 2:55 PM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] S-S in EC Dear All, I need to express a mammalian protein with S-S bridges and would hope to talk off-board with a person who knows about thioredoxin strains ect that might work. Thx, br ----------------------------------------------------------------- Bernhard Rupp 001 (925) 209-7429 +43 (676) 571-0536 [EMAIL PROTECTED] [EMAIL PROTECTED] http://www.ruppweb.org/ ----------------------------------------------------------------- The hard part about playing chicken is to know when to flinch -----------------------------------------------------------------
