WT Bsub_RlmN and evolved variants were expressed, purified and reconstituted for their iron-sulfur clusters using modified versions of previously published protocols (18 (link),34 (link),35 (link)). Briefly, enzymes were overexpressed and purified by Talon chromatography (Clontech). After chemical reconstitution of the iron-sulfur cluster, proteins were further purified by FPLC either on a Superdex 200 10/30 column or on a HiLoad 26/60 Superdex 75 Prep grade column (GE Healthcare Life Sciences) using 10 mM HEPES (pH 7.5) buffer containing 500 mM NaCl, 10% glycerol and 5 mM DTT. The fractions containing protein were combined and concentrated before being stored at −80°C.
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Purification and Reconstitution of Iron-Sulfur Enzymes
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Corresponding Organization : University of California, San Francisco
Other organizations : Weizmann Institute of Science
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Variable analysis
independent variables
- WT Bsub_RlmN and evolved variants
- Iron-sulfur cluster reconstitution
- Purification using Talon chromatography (Clontech)
- Chemical reconstitution of the iron-sulfur cluster
- Further purification by FPLC on Superdex 200 10/30 column or HiLoad 26/60 Superdex 75 Prep grade column (GE Healthcare Life Sciences)
- 10 mM HEPES (pH 7.5) buffer containing 500 mM NaCl, 10% glycerol and 5 mM DTT
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