protein solubility problems

Malcolm M. Campbell malcolm.campbell at plants.ox.ac.uk
Thu Feb 20 09:52:31 EST 1997


We are working with a recombinant plant protein that is a myb
transcription factor. We have the protein expressed in E. coli (using
Novagen pET-30c vector), and are attempting to purify it using
Ni-affinity, then cut the tags off with enterokinase or thrombin, and
finally run crystallization trials for eventual x-ray crystallography. 
The protein is highly expressed, and is mostly (>90%) in the bacterial
pellet, probably in inclusion bodies and/or complexed with nucleic acids. 
I can purify (Ni-affinity) 5 mg or more from a 100 mL culture if I use
denaturing conditions (urea) and elute with low pH.  With non-denaturing
conditions (Na-phosphate buffer w/ sonication), I get ~0.2 mg from 100 mL
culture.  The imidazole elution does not seem to purify the protein as
well as pH in urea- there are numerous other bands.

My questions are these:
1. Since we want to use the protein for x-ray crystallography, is it
necessary to conduct purification, etc, under non-denaturing conditions?

2. Is there a method for getting more of the protein out in the soluble
fraction, or removing it from the insoluble fraction while maintaining it
in non-denaturing conditions?
I have an article by Hoess, et al (Bio/technology 6:1214, 1988) describing
recovery of soluble recombinant proteins from inclusion bodies using
Q-sepharose ion exchange resin.  Is this method, or another one, commonly
used?

Thanks for any advice anyone can give.
Nancy A. Eckardt
Postdoctoral Researcher
University of Oxford
Department of Plant Sciences

-- 
Malcolm Campbell                 malcolm.campbell at plants.ox.ac.uk
Fibre Biotechnology Lab          44.1865.275135 (voice)
Department of Plant Sciences     44.1865.275074 (FAX)
University of Oxford
Oxford OX1 3RB, UK



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