Have you tried adding a detergent such as SDS to your cells when you lyse them? This in my experience helps improve the soluble fraction significantly although it may lead to downstream problems with removal of the detergent.
From: xaravich ivan [mailto:[email protected]] Sent: 17 October 2014 02:52 PM To: [email protected] Subject: [ccp4bb] Strategies to bring out over-expressed protein from inclusion bodies to soluble fraction!!! Dear cc4bb enthusiasts, This is slightly off topic but many protein crystallographers might be familiar with this problem. I have been trying to over-express a bacterial (non-E.Coli) protein in E.Coli and more than 80% goest to inclusion bodies. I tried the following Lowering the IPTG concentration to 0.1mM (range test from 0.075 mM - 0.5 mM) Cold shock for 30 minutes in ice before induction Slow rotation speed at 18 degrees O/N after induction While these steps helped a bit I still get about 50-60% of my protein in inclusion bodies. I would like to know what other steps do you suggest to enhance the yield in the soluble fraction (without changing the host strain or manipulating the DNA) Thanks in advance Ivan <table width="100%" border="0" cellspacing="0" cellpadding="0" style="width:100%;"> <tr> <td align="left" style="text-align:justify;"><font face="arial,sans-serif" size="1" color="#999999"><span style="font-size:11px;">This communication is intended for the addressee only. It is confidential. If you have received this communication in error, please notify us immediately and destroy the original message. You may not copy or disseminate this communication without the permission of the University. Only authorised signatories are competent to enter into agreements on behalf of the University and recipients are thus advised that the content of this message may not be legally binding on the University and may contain the personal views and opinions of the author, which are not necessarily the views and opinions of The University of the Witwatersrand, Johannesburg. All agreements between the University and outsiders are subject to South African Law unless the University agrees in writing to the contrary. </span></font></td> </tr> </table>
