Cloning, Expression, and Purification of the Nitrite Reductase Gene from Lactobacillus plantarum DMDL 9010
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Abstract:
The nitrate reductase gene (nir), encoding the nitrite reductase enzyme (NiR) in Lactobacillus plantarum DMDL 9010, was cloned, and the expressed product was purified. The nir fragment was amplified by polymerase chain reaction (PCR) using L. plantarum DMDL 9010 DNA as template. Sequencing studies revealed that the nir fragment was 1638 bp in size. The gene was cloned into plasmid pET-32a(+) and was transferred into competent Escherichia coli DH5α cells in order to construct the pET-32a(+)-nir-DH5α recombinant plasmid. pET-32a(+)-nir was transferred into the expression strain E. coli BL21 to construct the denitrification recombinant bacterium, pET-32a(+)-nir-BL21. The recombinant bacterium was induced by 1 mmol/L IPTG to produce NiR protein when the induction temperature was maintained at 30 ℃ for 4 h, and more than 90% of NaNO2 (50 ?g/ml) was degraded. High-purity NiR was obtained by His-Tag nickel affinity chromatography (Ni Sepharose 6 Fast Flow), and the molecular weight of NiR, as measured by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), was approximately 60 kDa. In summary, nir from L. plantarum DMDL 9010 was successfully cloned, and the recombinant bacterium pET-32a (+)-nir-BL21 effectively degraded nitrite. High-purity NiR can be obtained by affinity chromatography.
