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Screening and purification for novel cytochrome b 5 from uncultured environmental micro‐organisms
Author(s) -
Roh C.,
Villatte F.,
Kim B.G.,
Schmid R.D.
Publication year - 2007
Publication title -
letters in applied microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.698
H-Index - 110
eISSN - 1472-765X
pISSN - 0266-8254
DOI - 10.1111/j.1472-765x.2007.02118.x
Subject(s) - bacteria , biology , microbiology and biotechnology , chemistry , computational biology , genetics
Abstract Aims: We describe a sequence‐based PCR method suitable for the isolation of a novel soluble heme‐binding domain of cytochrome b 5 (cyt b 5 ) gene directly from metagenomic DNA is described. Methods and Results: Using the degenerate primer set, a cyt b 5 gene was isolated directly from metagenomic DNA. Based on the sequence‐based PCR method, the similar conserved motif of cyt b 5 from Rhodopseudomonas palustris strain makes the novel target gene. The gene encoding cyt b 5 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.