Hi Jan, How is the Fe-S cluster deformed? What is the evidence? Has your colleague done Fe and S analysis? There is a lot of missing information to this question that prevents a much more thorough answer. Is the protein membrane associated? Part of a larger complex? Besides the Fe-S cluster there are a number of other things that can be an issue. However, many Fe-S clusters are sensitive to aerobic conditions. If your colleague's cluster is structural, then when it degrades under aerobic conditions this can lead to protein aggregation or instability. How well does the protein express? It is also possible during overexpression to out pace the Isc machinery of the E. coli cell and thus not have full integration of your Fe-S cluster, which again could lead to aggregation. I think the solutions to either of these scenarios are obvious and may require a collaborator to really determine the best way to pursue this protein purification. As has been mentioned an exogenous Isc operon plasmid can be used to mitigate the effects of the later situation, but is not the only solution.
Good luck, Chris -- Christopher L. Colbert, Ph.D. Assistant Professor Department of Chemistry and Biochemistry North Dakota State University P.O. Box 6050 Dept. 2710 Fargo, ND 58108-6050 PH: (701) 231-7946 FAX: (701) 231-8324 From: Jan Rashid Umar <jan...@googlemail.com<mailto:jan...@googlemail.com>> Reply-To: Jan Rashid Umar <jan...@googlemail.com<mailto:jan...@googlemail.com>> To: "CCP4BB@JISCMAIL.AC.UK<mailto:CCP4BB@JISCMAIL.AC.UK>" <CCP4BB@JISCMAIL.AC.UK<mailto:CCP4BB@JISCMAIL.AC.UK>> Subject: [ccp4bb] FE-Sulfur proteins Dear All, I will be grateful to your suggestions about Iron-Sulfur cluster protein purification. My colleague has some problems with the purification and it seems that the iron-sulfur cluster might be deformed, and protein is aggregated. The purification is done under aerobic (normal condition), and is there some method to reintegrate iron sulfur cluster back into the protein molecule under these conditions. Can anybody suggest some literature, protocol or something that can improve the protein aggregation? I look forward to hearing from you. Best wishes, Jan
