Formate dehydrogenase from methylotrophic bacterium Pseudomonas sp. 101: gene cloning and expression in Escherichia coli.

Abstract

Using two degenerate 20- and 23-mer oligonucleotide probes, the gene encoding NAD(+)-dependent formate dehydrogenase (FDH) (EC 1.2.1.2) was shown to lie within a 3.5 kb PstI fragment of the chromosomal DNA of methylotrophic bacterium Pseudomonas sp. 101. A phasmid library was prepared in the lambda pSL5 vector with partial EcoRI-digested DNA from Pseudomonas sp. 101. The 12 clones selected contained three types of phasmid: lambda pFDH1, lambda pFDH2 and lambda pFDH3 (with inserts of 15, 17.6 and 18.5 kb respectively). The inserts contained the same 15 kb EcoRI and 3.5 kb PstI fragments and included the complete FDH gene. Further subcloning of the insert resulted in plasmid pFDH2 and a 2.32 kb HindIII-BgIII fragment. Active enzyme was expressed in Escherichia coli TG1 (pFDH2) strain under control of a lac promoter.