Insoluble Protein

iwilson at molbiol.ox.ac.uk iwilson at molbiol.ox.ac.uk
Mon Oct 25 13:06:32 EST 1993


In article <18OCT199313510964 at mozart.biosci.arizona.edu>, xiong at mozart.biosci.arizona.edu (Zhongguo Xiong) writes:
> Dear Netters:
> 
> I have recently encountered a insoluble recombinant protein and need all kinds
> of suggestions or comments I can get. I over-expressed a plant RNA viral
> protein (possible a RNA-depedent RNA polymerase protein) in E. coli. The
> protein formed an inclusion body in the bacteria. I had no problems in
> solublizing the protein with urea and the subsequent protein purification in
> the nickel column. Then I dialyzed the purified protein against a series of
> Urea buffers with decreasing concentrations of Urea (from 8M to 0). Inspite of
> the precaution, the protein still formed precipitates. I tried to resolublize
> the protein in 4 M urea and dit not see any protein in SDS PAGE gel.
> 
> Are there any way to make the protein soluble in a non-denaturing condition?
> Any comments and suggestions are welcome.> 
> Z. Xiong

In our group someone experimented with adding Cu(II) ions - I don't have the 
details with me and I have also recently seen a paper on oxido-shufling with 
glutathione GSH + GSSG mixtures since disulphides were involved (see recent 
paper in Protein Engineering on galactosyltransferase.

Iain Wilson
Dyson Perrins Laboratory, University of Oxford iwilson at molbiol.ox.ac.uk



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