Purification and Activity of Human Peroxiredoxin 1 Expressed in E.coli
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Abstract:
In order to obtain active human peroxiredoxin 1(Prx1) with high purity, Prx1 cDNA sequence was amplified by polymerase chain reaction (PCR), cloned in expression vector pET28 to construct a prokaryotic expression plasmid pET28-Prx1, containing full-length human Prx1 gene. After endonuclease digestion and DNA sequencing, the plasmid was transferred into Escherichia coli BL21 (DE3) for expression. The expression product was identified by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and western blotting. The recombinant protein was purified by nickel-nitrilotriacetic acid (Ni-NTA) affinity chromatography, the Michaelis constant (Km) for Prx1 was determined, and the activity of Prx1 was identified by plasmid protection assay. The result of endonuclease digestion and DNA sequencing showed that recombinant plasmid was constructed successfully. The expression product was approximately 25 kDa, as determined by SDS-PAGE and western blotting, which is consistent with the actual molecular weight of human Prx1. After purification, the yield of Prx1 was 7.59 mg/g bacteria, with a purity of 88.50%. The Km value of purified Prx1 was 2.06×10-4 mol/L in catalytic reduction of H2O2 at 37 ℃ with a pH value of 7.4. Plasmid protection assay showed that Prx1 could protect pUC18 DNA from oxidative damage. Thus, Prx1 was successfully expressed in E.coli and an active protein with high purity was obtained.
