人对氧磷酶1在巴斯德毕赤酵母中的表达与纯化

Expression and Purification of Human Paraoxonase 1 in Pichia pastoris

摘要:

人对氧磷酶1（Human Paraoxonase 1，hPON1）是人体血液中的一种非专一性酯酶，可以水解多种有机磷化合物，被认为是一种有机磷农药中毒的解毒剂。为了得到较纯的具有活性的hPON1，本文采用巴斯德毕赤酵母Pichia pastoris表达系统，对hPON1进行胞内表达。hPON1基因经过密码子优化后克隆至pPICZA表达载体上，构建得到pPICZA-PON1重组表达质粒，经线性化后转化至巴斯德毕赤酵母X33菌株中，筛选得到阳性重组菌株。将重组菌株进行摇瓶发酵120 h，酶活达0.15 U/mL。收集发酵液并细胞裂解后，用Ni-NTA螯合亲和层析的方法进行纯化，得到纯化的hPON1，SDS-PAGE结果显示表达产物的大小约37 ku，与预期的蛋白分子量相符。表达的重组hPON1的最适反应温度为45 ℃，最适pH为9.0。以上结果表明，本研究成功地表达并得到较纯的有活性的hPON1蛋白。

Abstract:

Human paraoxonase 1 (hPON1) is a non-specific esterase in human plasma that can hydrolyze various organophosphates, and is considered as an organophosphate pesticide scavenger. In order to obtain high-purity active hPON1, a Pichia pastoris expression system was used to conduct intracellular expression of hPON1. The hPON1 gene was codon-optimized and cloned into the pPICZA expression vector to obtain the recombinant plasmid pPICZA-PON1. The recombinant vector was linearized and transformed into Pichia pastoris X33, and positive transformants were verified by colony polymerase chain reaction (PCR). Batch fermentation of the recombinant strains was performed in shake flasks for 120 h, and the esterase activity of hPON1 was 0.15 U/mL. Cells were collected and lysed and the recombinant protein was purified by nickel-nitrilotriacetic acid (Ni-NTA) affinity chromatography. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) results showed that the molecular mass of the recombinant hPON1 was approximately 37 ku, consistent with the expected value. The optimal reaction temperature and pH for the expression of recombinant hPON1 were 45 ℃ and 9.0, respectively. The above results demonstrated that active recombinant hPON1 was successfully expressed, and high purity and active hPON1 was obtained in this study.