Hi Rafael, We have had great success across many projects using Arctic Express with induction at as low as 4C, often getting high level soluble expression rather than insoluble lumps. https://www.agilent.com/en/product/protein-expression/competent-cells-for-difficult-protein-expression/insoluble-proteins/arcticexpress-%28de3%29-competent-cells-232939
We have also generally found that we get much better recovery of ‘difficult’ proteins using a cell disruptor rather than sonication or chemical lysis (we use a continuous flow cell disruptor from constant systems) Haven’t delved into this in detail, but do wonder if this paper provides some of the reason: https://pmc.ncbi.nlm.nih.gov/articles/PMC4388321/ Good luck! Take care, Charlie. From: CCP4 bulletin board <CCP4BB@JISCMAIL.AC.UK> On Behalf Of Rafael Marques Sent: 11 March 2025 11:19 To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] [OFF-Topic] GST-tagged protein refold protocol WARNING: This message was sent by an external party. Report suspicious messages via the “Report Phishing” button in Outlook. 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 S i This message needs your attention * No employee in your company has ever replied to this person. * This is a personal email address. Powered by Mimecast CGBANNERINDICATOR 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<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/
