Dear Umar,
Have you determined the Fe-S cluster content of the as-isolated protein? If you use common BL21 cells for protein production, the protein is most likely not fully maturated (the majority of the protein does probably not contain a Fe-S cluster). Thus, you may have free Cys (if the cluster is coordinated by Cys, which is the most common Fe-S ligand) in the protein and free thiol groups on the GST beads, which may cross-link or have any other interaction. The critical step is really to have a fully assembled Fe-S cluster in most cases, thus, I would recommend to maturate the protein using chemical reconstitution or using a cell line that is specifically tailored for Fe-S protein production. You could also design another construct, using e.g. Strep tag. I would avoid immobilized metal affinity chromatography, since you may end up with metal impurities in the protein. Good luck! Ingrid From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Umar Farook Sent: Saturday, June 27, 2020 6:09 PM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Looking for suggestions with protein expression Glutathione beads for GST tagged protein. Umar On Sat, 27 Jun 2020, 6:56 pm John Newitt, <newit...@gmail.com <mailto:newit...@gmail.com> > wrote: On Jun 27, 2020, at 3:15 AM, Umar Farook <umarfaroo...@gmail.com <mailto:umarfaroo...@gmail.com> > wrote: > > > Dear All, > > Sorry for an offtopic question, your suggestions are highly appreciated. > > We have been working on iron sulfur cluster binding protein, which is usually > expressed as a nice soluble protein expressed in BL21 cells but aggregated in > the affinity column itself and unable to recover from it. We had made n > number of truncations and fused to soluble tags such as MBP, but always ended > up in large aggregates. Anyone has experience in working with iron-sulfur > cluster binding protein before, please let us know the critical steps in > purification of such proteins, whether you have completely done the > expression, purification and crystallization in anaerobic conditions? or else > changing the expression system to eukaryotic system such as Baculo or HEK > 293T would help? > > Please share your valuable experience, thank you. > > When you say “affinity column”, are you referring to a Ni2+-affinity column? -John _____ To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB <https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB&A=1> &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/
