Dear Dipankar, Your problem is quite tricky to solve. I have two opinions which worked for me for a very high aggregation prone protein (Huntingtin, which always used to go in inclusion bodies and precipitate even in elution buffer). 1. First have 4-5M Guanidinum Hydrochloride in the lysis buffer during the purification process, and keep 150mM proline in the elution buffer. I have experienced that addition of proline greatly reduces the aggregation propensity. 2. Second option is dealing the problem with advantage of that problem. Try to precipitate the protein (which is in elution buffer) with acetone (soon after elution) and resolubilize it in your desired final buffer very rapidly under cooling conditions (like in 4 degree centigrade). Though the 100% protein may not resolubilize, but it will fairly solubilize as maximum as up to 90% (as that happened with me). My intuition is that in the final solubilized fraction you will get high proportion of folded form of your protein. Just check if it works for you.
Best wishes!! Debasish CSIR- Senior Research Fellow C/o: Dr. Akash Ranjan Computational and Functional Genomics Group Centre for DNA Fingerprinting and Diagnostics Hyderabad, INDIA Email(s): dkgh...@cdfd.org.in, dgho...@gmail.com Telephone: 0091-9088334375 (M), 0091-40-24749396 (Lab) Lab URL: http://www.cdfd.org.in/labpages/computational_functional_genomics.html ----- Original Message ----- From: "Dipankar Manna" <dipankar.biot...@gmail.com> To: CCP4BB@JISCMAIL.AC.UK Sent: Thursday, September 21, 2017 3:42:28 PM Subject: [ccp4bb] Precipitation issue during refolding 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 E-mail: dipankar.ma...@medisin.uio.no dipankar.biot...@gmail.com http://www.med.uio.no/imb/english/people/aca/dipankam/
