I am purifying different proteins that go into inclusion bodies when expressed in E. coli. When I purify my inclusion bodies I always find two bands around 37 kDa in my solubilized fraction. These proteins are partially washed off with 20 mM Tris pH 8, 2 M urea (lane 1 and 2 in my gel picture), but I have the protein also in the solubilized fraction (lane 3). Some of the contaminant protein precipitates in the refolding step (lane 4 shows soluble and refolded protein) but a low amount of these proteins are still present. I have problems getting rid of these proteins in downstream purification steps. I therefore wonder whether anyone here on ResearchGate knows the identity of this protein/ these proteins, so that I can design a purification protocol that will separate my target protein from these E. coli contaminants.
My inclusion body purification protocol is:
1. Resuspending the insoluble fraction after bacterial lysis in 20 mM Tris pH 8, 500 mM NaCl, 2 M urea. Spin down and repeat this step.
2. Solubilization (20 mM glycine pH 10, 6 M urea, 5 mM B-Me)
3. Refolding
Your washing procedure is mild, use the protocol described here (Article Production and Immunogenicity Assessment of LTp50: An Escher...
), hopefully these proteins will be washed away. Still, you must run chromatography to get a purer recombinant protein.
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