Purification and Reconstitution of NrdI and NrdF

Chemical
reagents were obtained from Sigma-Aldrich
at the highest purity available. Restriction enzymes, T4 DNA ligase,
and isopropyl-β-d-1-thiogalactoside (IPTG) were purchased
from Promega (Madison, WI). All DNA sequencing of cloned genes was
carried out at the Massachusetts Institute of Technology Biopolymers
Laboratory. Ni-NTA Fast Flow affinity resin was from Qiagen. Complete
EDTA-free protease inhibitor tablets and calf alkaline phosphatase
(20 μmol min^–1 μL^–1) were purchased from Roche Biochemicals (Indianapolis, IN). Amicon
Ultra-15 centrifugal filter devices were from Millipore. N-Terminally
His₆-tagged NrdI and apo-NrdF were expressed and purified
as reported previously.^{9 (link),27 (link)} Apo-NrdF was reconstituted with
Mn^III₂-Y· and Fe^III₂-Y· as previously described.^{9 (link),28 (link)}