人抗氧化蛋白-1在大肠杆菌中的表达、纯化及活性鉴定

Purification and Activity of Human Peroxiredoxin 1 Expressed in E.coli

摘要:

为得到较纯的具有活性的人抗氧化蛋白Peroxiredoxin1（Prx1），本文先用PCR的方法扩增了人Prx1的cDNA序列，然后将其连接到pET28表达载体上，从而构建了编码全长的人抗氧化蛋白-1基因的pET28-Prx1原核表达质粒。经双酶切及测序鉴定后将表达质粒转入大肠杆菌BL21（DE3）进行表达，并通过SDS-PAGE和western blot来鉴定表达产物。随后用Ni-NTA螯合亲和层析的方法纯化重组蛋白，并测定该酶的米氏常数，用质粒保护实验鉴定该蛋白其活性。最终表达载体经酶切和测序鉴定证实构建成功，表达产物在SDS-PAGE和western blot图的25 kD左右，与prx1真实的蛋白分子量相符，说明Prx1蛋白成功表达。Prx1纯化后产量达到7.59 mg/g菌体，纯度达到88.50%。纯化后的Prx1在37 ℃和pH 7.4的条件下催化H2O2反应的米氏常数Km为2.06×10-4 mol/L,质粒保护实验表明Prx1可以保护pUC18质粒免受氧化损伤。因此，Prx1在大肠杆菌中成功表达并得到较纯的活性蛋白。

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.