Human Sigma 1 Receptor Expression

Steve Hobbs hobbs at icr.ac.uk
Mon Mar 18 09:40:15 EST 2002


Hello All,

I've been trying to express a human sigma 1 receptor cDNA
(EMBL:HSU75283) in E. coli as a C-terminal 6xHis fusion protein using
vectors pET-24b(+) (NdeI/XhoI insert) and pET-24d(+) (NcoI/XhoI
insert).  So far I've tried the standard BL21(DE3)pLysS strain and
variants BL21 CodonPlus-RP (Stratagene, for correcting Arg and Pro codon
bias) and BL21 STAR(DE3)pLysS (Invitrogen, RNAseE mutant for increased
expression).  The ORF should give a protein of approximately 26kD, but I
cannot see any expression by Coomassie Blue staining on induction with
1mM IPTG, either overnight at 37C or over a time course of 0-5hr at 37C,
neither in the cell lysate nor pellet (inclusion body) fraction after
lysis.  All the usual controls are fine, test protein expressing well
etc, but the induced lanes look identical to the uninduced and empty
vector lanes.  Cells were lysed by Novagen BugBuster reagent + Benzonase
(to reduce DNA viscosity) + protease inhibitor cocktail.  I need a
reasonable amont of protein to purify for immunisation so need good
expression, i.e. visible by CB staining, so if it's only visible by
western blotting I'm not interested.

Any thoughts?  The initial part of the ORF (40 or so bp) is GC-rich but
no adverse bias codons are present.  Could this affect expression in E.
coli?

Many thanks,

Steve Hobbs



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