Over-expression and Purification of Formate Dehydrogenase form Pichia pastoris in Escherichia coli

Abstract:

Formate dehydrogenase(FDH)coding gene was amplified from genomic DNA of Pichia pastoris by polymerase chain reaction, and the codon TAG(bases 649-651)was mutated to GAG using site-directed mutagenesis.The recombinant plasmid pET-FDH was con- structed by inserting the mutated DNA fragment into expression vector pET-22b(+),and transformed into E.coli BL21(DE3).FDH was expressed as a form of soluble prutein fused with 6×His tag at high level through IPTG induction.The amount of FDH was up to about 30% of the total cell protein. The cells-free crude extract was purified by one affinity chromatographic step,and resulting enzyme preparation revealed a specific activity of 6.45U/mg.