problems renaturing recombinant protein

Sarah Tilley sjt at newt.phys.unsw.edu.au
Wed Jul 7 02:46:46 EST 1993


--
In article <kcrossgr-260693131802 at gradmac.bio.upenn.edu>, kcrossgr at mail.sas.upenn.edu (Kirsten Crossgrove) writes:
|>
|>Hi everyone,
|>I'm trying to express a zinc-finger containing protein in E. coli.  It
|>expresses well but is very insoluble.  I can solubilize it in 6M guanidine
|>HCl and purify it over a nickel-chelate column (it has a 6 histidine
|>N-terminal fusion).  However, when I dialyze to get rid of the GuHCl (or 8M
|>urea-same result), the protein sometimes precipitates out, and even when it
|>doesn't, the activity is extremely low to non-existent.  The protein is
|>eluted off the column at low pH (usu. 5.9-6.3).  The dialysis buffer is:
|>10mM HEPES pH 7.9
|>80mM KCl
|>1mM DTT
|>0.1% Triton X100
|>5-20% glycerol
|>10uM ZnCl2
|>I get rid of the GuHCl or urea in a stepwise manner (1M, 0.1M, 0M).
|>I've gotten reasonably active preps before, but it is just not consistent
|>and lately no matter what I do, I can't get active protein. I think it is a
|>problem with the renaturation. 
|>Any suggestions?
|>Thanks.
|>Kirsten Crossgrove
|>

Have you tried the E.coli chaperonin60, GroEL? It has been suggested in some recent articles, that such chaperonins might need to be co-expressed when trying to produce active recombinant proteins.
**
Sarah Tilley
sjt at newt.phys.unsw.edu.au



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