Dear Rafael, do you know if your protein interacts with another protein? This could be critical. I have been studying proteins that require a protein partner to be expressed as soluble protein. Co-expressing both proteins in E. coli shifts the expression from insoluble to soluble.
Hope this helps. Best wishes, Marc — Marc GRAILLE, PhD DR1-CNRS Head of the team: “Translation and degradation of eukaryotic mRNAs” Laboratoire de Biologie Structurale de la Cellule (BIOC); UMR7654; CNRS ÉCOLE POLYTECHNIQUE 91128 PALAISEAU CEDEX FRANCE 📞: +33 (0)1 69 33 48 90 https://portail.polytechnique.edu/bioc/en/research/coupling-between-translation-and-mrna-degradation-eukaryotes Responses to emails are not expected outside of your normal working hours.  > Le 13 mars 2025 à 18:14, Rafael Marques <rafael_mmsi...@hotmail.com> a écrit : > > Hi Dave, > > I should have mentioned that, but yes, I tried 16 degrees overnight and even > up to 40 hours. The expression level is great, majority is in the pellet > though and the elution fraction is far from desirable for my kinetic assays. > > Best wishes > > ______________________________________________________ > > > 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" > > > On 11 Mar 2025 13:16, David Briggs <david.bri...@crick.ac.uk> wrote: > 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 > ######################################################################## 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/
