Isotopic DHFR Preparation for NMR

Reduced β-nicotinamide adenine
dinucleotide phosphate tetrasodium salt hydrate (NADPH), d-glucose-6-phosphate sodium salt, and glucose-6-phosphate dehydrogenase
from Leuconostoc mesenteroides were
purchased from Sigma-Aldrich. (6S)-5,6,7,8-Tetrahydrofolic
acid (THF) was obtained from Schircks Laboratories. Expression and
purification of L28F DHFR were performed as described previously.^{6 (link),26 (link)} Isotopically labeled L28F DHFR for proton relaxation dispersion
measurements was expressed in M9 medium containing 0.5 g/L of ¹⁵NH₄Cl, 0.5 g/L ¹⁵NH₄(SO₄)₂, and 3 g/L [¹³C,¹H]-glucose
in 99% D₂O according to published protocols.^{27 (link),28 (link)}