If you want to do anything with iron-sulfur proteins you need to work in strict anaerobic conditions as these are extremely sensitive to oxygen (99% of the time).

If you want to give a go at purification of denatured protein, here is a good protocol to follow for his-tagged proteins (page 17: http://tools.thermofisher.com/content/sfs/manuals/ninta_system_man.pdf).

For a simple reconstitution protocol can look at this paper (https://www.jbc.org/article/S0021-9258(17)47056-6/pdf), however reconstitution rarely works for Fe-S proteins, but you can get lucky and again this requires a specialist anaerobic cabinet (not one used for bacterial culture, but where oxygen concentration is at ppm). In one of the labs I worked over the years they tried to reconstitute around 10 different proteins with only one actually working out.

thanks Rokas.. i am giving it a try now.. Lets see. I have another question, after refolding, does the protein reconstitute Fe S cluster naturally? or reconstitution of clusters in anaerobic chamber is a necessity

Hi,

Did you try to induce your protein at low temperature 16-18 0C? Also, you can try other E. coli strains as pLysS, BL21-AI or Origami™ 2 (https://blog.addgene.org/plasmids-101-e-coli-strains-for-protein-expression) and fuse SUMO or MBP proteins (vectors pET-Sumo and pMAL, respectively) in the N-terminal of your iron-sulfur protein to increase its solubility. I worked with a zinc finger protein and I got it soluble fusing it in MPB and NusA. See the manuscript 10.1105/tpc.20.00297. Good luck!

Hi Maxuel, the protein is tagged with SUMO, the only tag I am thinking of is GST, might be His and Fe bind and form comples, hypothetically, the strain is already plysS Rosetta, doesnt express in normal BL21, needs codon optimization, so I think those conditions arent much to explore..Any suggestions?Plus it is a 100kDa protein, so bigger tags might not be a solution

Hi Shreyoshi,

Did you try to induce your protein in a low temperature 16-18 0C? Try it with Rosetta strain. Sometimes for me MBP fusion works better than SUMO or GST. Maybe you can try MBP also (145 kDa is fine to induce in E. coli). All the best!