Hi Jan,
I wonder if the protein has a hexaHis tag. I recently was working on a Fe-S
containing protein and noticed significant aggregation/precipitation. After I
cleaving the His tag, the enzyme seems stable for days in the same buffer.
HTH,
Yuri
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),
Jan,
Do you express the protein with the *E. coli isc* iron-sulfur cluster synthetic
operon?
I found great success, see:
Daughtry, KD et. al. JACS 2012
http://pubs.acs.org/doi/full/10.1021/ja2111898
- Kelly Daughtry
***
Kelly Daughtry, Ph.D.
Good to know about this ISC operon!
Do you know if it is specific for Rieske-type His2/Cys2 Fe2S2 clusters,
or for FeS clusters in general?
Thus wild-type E. coli is already making three types of ISC clusters
for succinate dehydrogenase or fumarate reductase (although some help
might be needed
I *believe* that the ISC operon is utilized in E. coli for all FeS
clusters, regardless if Rieske or not.
I know that when deleted, activity of FeS containing proteins (succinate
dehydrogenase and Glutamate synthase) greatly decreases. See:
http://www.ncbi.nlm.nih.gov/pubmed/11432781
Thus it
To: CCP4BB@JISCMAIL.AC.UKmailto:CCP4BB@JISCMAIL.AC.UK
CCP4BB@JISCMAIL.AC.UKmailto: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
Have you looked at a UV-vis scan, there are some characteristic bumps you
should see depending on the state of your FeS cluster, plus you can monitor if
oxidation occurs over time. If I remember it correctly 322 nm is where you want
to look at.
Jürgen
On Jul 11, 2012, at 6:22 AM, Jan Rashid
