Hi,
I am working with a serine protease. As the protein is not soluble I am
purifying it from the inclusion bodies followed by refolding. The protein
shows good activity after refolding but the major concern is the
'precipitation'. Though the protein express quite well, but I loose almost
50-60% protein during refolding (Buffer: 100 mM Tris pH 8.0, 125 mM MgCl2,
100 mM KCL, 5% Glycerol, 2 mM GSH, 1 mM GSSG and 1 M NDSB) as it
precipitates. Refolding is done in room temperature. I tried refolding at 4
degree, but it even end up with more precipitant. It also precipitates
during concentrating, so in general I almost loose most of the protein
during this refolding and concentration steps. I start with 6 lit culture
that give around 1-2 mg protein in the final step, which I am not happy
with.
​
Any suggestion to deal this issue would be highly appreciated.
Thank you in advance.
Best,
Dipankar​
--
*Dipankar Manna, Ph.D*
Postdoctoral Researcher
Department of Molecular Medicine
Institute of Basic Medical Sciences
University of Oslo, Domus Medica
Oslo, Norway
Mob : +47 451 66 517 <451%2066%20517>
E-mail: dipankar.ma...@medisin.uio.no <dipankar.ma...@kjemi.uio.no>
dipankar.biot...@gmail.com
http://www.med.uio.no/imb/english/people/aca/dipankam/
