An improved method for the expression and purification of porcine dihydropyrimidine dehydrogenase.

摘要: Abstract Dihydropyrimidine dehydrogenase (DPD) catalyzes the reduction of uracil and thymine bases with electrons derived from NADPH. The mammalian DPD enzyme is a functional homodimer has an elaborate cofactor arrangement. Two flavin cofactors (FAD FMN) reside in two active site cavities that are separated by around 60 A. flavins apparently bridged four Fe4S4 clusters, which provided partner protomer dimer. study been hampered modest yield both native sources heterologous expression E. coli. In addition, minimal obtained when gene fused to N-terminal 6His-tag. This limitation dictated use traditional purification methods made more challenging apparent over-expression truncated and/or non-active forms DPD. Here we detail result ~4-fold improvement porcine expressed coli BL21 DE3 cells via pET plasmid system. addition ferrous ions sulfate during induction provide small increase purified enzyme. However, FAD FMN cell lysis results substantial activity also reduces relative proportion non-active, high molecular weight protein contaminants. We describe permit correlation content amount thus simple, rapid quantitation evaluation sample.