Expression and antioxidant activity of HdhTPX2 gene from disk abalone Haliotis discus hannai Ino in yeast Pichia pastoris
QIAO Kun1， FANG Chun-hua1，2， CHEN Bei1， PENG Hui3， PAN Nan1，CAI Shui-lin1， XU Min1， CHEN Li-jiao2， HAO Hua3， LIU Zhi-yu1
1. Key Laboratory of Cultivation and High-value Utilization of Marine Organisms in Fujian Province, Fisheries Research Institute of Fujian, Xiamen 361013,China; 2.School of Food Science，Fujian Agriculture and Forestry University, Fuzhou 350002, China; 3.College of Ocean and Earth Sciences, Xiamen University, Xiamen 361102, China
Abstract: HdhTPX2 gene was cloned into pPIC9K vector and transformed into yeast Pichiapastoris GS115 strain by electroporation to obtain recombinant expression plasmid pPIC9K-HdhTPX2 in order to study the antioxidant activity of HdhTPX2 from disk abalone Haliotisdiscushannai Ino. The recombinant product was induced by methanol, purified using immobilized metal affinity chromatography and confirmed using MS-fingerprinting to gain more information on its antioxidant function. The recombinant plasmid GS115/pPIC9K-HdhTPX2 was shown to be constructed successfully, and the optimal expression was found under conditions of incubation with 0.5% methanol for 24 h at 28 ℃ and pH 6.0, with a stable expressed product with molecular weight of about 25 000, consistent with the calculated molecular weight of HdhTPX2. The mass spectrum analysis revealed that the recobinant HdhTPX2 as purified recombinant protein showed stronger ability to scavenge hydroxyl radicals than VC and protect cells against the toxicity caused by H2O2 in vitro antioxidant activity. The findings indicate that yeast P.pastoris is an effective expression system for producing large quantities of biological active HdhTPX2.
2019, Vol. 34 
