Refolding protocol is often specific to the protein. As a rule of thumb, consider exploring refolding in large dilution regime as well as dialysis and on-column methods. As far as refolding GST fusions, I personally recommend re-cloning without GST and refolding the guest proteins by itself. This considerably improves the odds.
Artem On Tue, Mar 11, 2025, 07:18 Rafael Marques <rafael_mmsi...@hotmail.com> wrote: > 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 > ######################################################################## 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/
