Hello Umar, I would not pin down your difficulties solely due to an Fe-S proteins. I have produced some with no fusion partners and they work wonderfully. They were expressed in an aerobic environment and then reduced in an anaerobic one before usage in reactions.
1) On the Fe-S side, there are plasmids you can co-transform to increase Fe-S production. This plasmid pH151 has the synthetic genes necessary for Fe-S formation. https://www.jbc.org/content/279/33/34721.abstract 2) On the general protein side, have you hhpred your protein? Different constructs (not just tags), temperature? Strain? Media? 3) For these proteins we typically use His Excel (or Protein Ark Ni2+ Advance) that is resistant to most chelators since more often than not, they contain other metals and can snatch Ni2+ from normal Ni-NTA resins. Strep tags also work well. On Jun 27, 2020, at 9:14 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. -- Best Regards, Umar Farook ________________________________ 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/
