Dear Rafael, I agree with the previous answers, decreasing the temperature to 16-18°C overnight during protein expression really helps with folding. It works wells with BL21(DE3) and Rosetta cells. I think there is even a E.coli strain for lower temperature but I have never used them myself.
Also, in our lab, we don’t use GST-tag but SUMO in some of our constructs but also a combination of either His6-SurA or His6-TriggerFactor to help solubilize the proteins. Careful with trigger factor, we experienced the protein following along our construct through SEC despite the tag removal step. In overall, if you can improve the solubility of your protein without rescuing them from inclusion bodies, it would be better, especially if you don’t know how to confirm the correct folding after this step (with activity assays). Good luck ! Marion Sent from my iPhone, please forgive any misspellings. Le 11 mars 2025 à 15:31, Artem Evdokimov <artem.evdoki...@gmail.com> a écrit : 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<mailto: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 ######################################################################## 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/
