Screening and purification for novel cytochrome b5 from uncultured environmental micro-organisms

Abstract

Aims: We describe a sequence-based PCR method suitable for the isolation of a novel soluble heme-binding domain of cytochrome b5 (cyt b 5) gene directly from metagenomic DNA is described. Methods and Results: Using the degenerate primer set, a cyt b5 gene was isolated directly from metagenomic DNA. Based on the sequence-based PCR method, the similar conserved motif of cyt b5 from Rhodopseudomonas palustris strain makes the novel target gene. The gene encoding cyt b5 was cloned and expressed in Escherichia coli BL21 (DE3) using pET expression system. The expressed recombinant enzyme was purified by Ni-nitrilotriacetic acid affinity chromatography and characterized. Conclusions: Sequence-based strategy is an effective method for application of the novel gene from metagenomic DNA. Significance and Impact of the Study: Investigation of novel genes from metagenome, most of the micro-organism species are largely untapped, could represent an interesting and useful reservoir for biological processes. © 2007 The Authors.