Hi Rafael, I have you tried reducing the temperature post-induction?
You can grow the cells to OD600 <whatever>, reduce the temperature (I have gone as low as 16ºC), add IPTG and harvest the cells the following morning. The reduction in temperature can slow down the rate of protein production allowing the nascent polypeptide chain more time to fold correctly. (At least this is how I rationalise the observed increased in folded soluble protein in my head) I would certainly play with the temperature before going down the refolding route. Good luck, Dave -- Dr David C. Briggs CSci MRSB (he/him) Principal Laboratory Research Scientist Signalling and Structural Biology Lab The Francis Crick Institute London, UK Working hours: Mon-Fri 0900-1700 == about.me/david_briggs<https://about.me/david_briggs> | OrcID<https://orcid.org/0000-0002-9793-7339> | Google Scholar <https://scholar.google.co.uk/citations?user=DRKG5KwAAAAJ> == "Would it not be better if one could really 'see' whether molecules...were just as experiments suggested?" – Dorothy Hodgkin ________________________________ From: CCP4 bulletin board <CCP4BB@JISCMAIL.AC.UK> on behalf of Rafael Marques <rafael_mmsi...@hotmail.com> Sent: 11 March 2025 11:18 To: CCP4BB@JISCMAIL.AC.UK <CCP4BB@JISCMAIL.AC.UK> Subject: [ccp4bb] [OFF-Topic] GST-tagged protein refold protocol External Sender: Use caution. Hello folks, I have been trying to purify a GST-tagged protein for a while but the results are far below the expected. The majority of my sample is in the pellet fraction and the yield of my expression is very good. This is known because the protein was identified both by Mass Spectrometry and Western-blot. I believe my protein is laying inside inclusion bodies. I have tried to diminish the IPTG concentration, express it for a shorter time and also different E.coli strains but the results are still the same. As a last resort, I was thinking about refolding. I found this paper (attached), from 2004, but I was wondering if someone who has experience regarding protein refolding could shed some light on this topic, principally if there are more updated protocols. Best wishes P.S. His-tag did not improve my results too ______________________________________________________ Rafael Marques da Silva PhD Student – Structural Biology University of Leicester Mestre em Física Biomolecular Universidade de São Paulo Bacharel em Ciências Biológicas Universidade Federal de São Carlos phone: +44 07861 273773 "A sorte acompanha uma mente bem treinada" ________________________________________________ ________________________________ To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB&A=1 The Francis Crick Institute Limited is a registered charity in England and Wales no. 1140062 and a company registered in England and Wales no. 06885462, with its registered office at 1 Midland Road London NW1 1AT ######################################################################## To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB&A=1 This message was issued to members of www.jiscmail.ac.uk/CCP4BB, a mailing list hosted by www.jiscmail.ac.uk, terms & conditions are available at https://www.jiscmail.ac.uk/policyandsecurity/
