Protein refolding

Shiverer shiverer at
Thu May 23 06:29:35 EST 1996

I am looking for some assistance in the refolding of a histagged protein 
that is expressed in E.coli.  The protein is an IgG like receptor and 
the expressed fragment is the extracellular domain which contains 12 
cysteine residues.  The protein is insoluble after expression and can be 
purified nicely under denaturing conditions.  The pure fractions are 
soluble in 8 M urea, however when I dialyze the urea away (even in he 
presence of reducing agents) the protein aggregates and precipitates.  I 
have tried diluting the protein, slow dialysis in the presence of redox 
system, 0.4 arginine etc to assist in refolding.  So far this protein 
aggregates at urea concentrations under 2 M.  Are there any suggestions 
as how to select cosolvents such as PEG or ethanol to help  in refolding 
?  Are there any good books that explain the use of salts, chaotropes, 
detergents, etc in a simple enough language that a molecular biologist 
would understand ?  The molecular biology cookbooks all declare that 
conditions have to be "determined empiricaly".  However there must be a 
logic behind the selection of buffer constituents.  For this protein I 
am not interested in obtaining the original structure (although the 
closer the better) but rather need a soluble fraction that can be used 
to coat plastic. Any help appreciated.  Could replies be sent to me 
directly at marsden at as I do not subscribe to this 
news group

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