关键词: 蒙古黄芪, 病程相关蛋白, 可溶性表达, 标签, 分子伴侣

Abstract: Astraglus membranaceus pathogenesis-related protein-10 (AmPR-10) has nuclease activity, which is of great significance to the growth and development of Astragalus membranaceus and its disease resistance mechanism. However, the traditional method of extracting protein from natural Astragalus membranaceus has higher cost and less protein yield. For the first time, Escherichia coli expression system was used to express AmPR-10, and three kinds of recombinants were constructed: ① pET28a-AmPR-10 constructed by pET28a; ② pET30a-AmPR-10 constructed by pET30a; ③ pET30a-skp-AmPR-10 constructed with the E. coli chaperone skp by pET30a. According to SDS-PAGE analysis, the soluble expression of target protein was pET30a-skp-AmPR-10 > pET30a-AmPR-10 > pET28a-AmPR-10. According to the simulation analysis of protein tertiary structure, the tag S-tag on the vector pET-30a could improve the soluble expression of the target protein by influencing the local α helix conformation of AmPR-10, and the molecular chaperone skp could further improve the soluble expression of the target protein by increasing the overall α helix ratio of the fusion protein. The study solved the problems of complex operation and small amount of protein extraction from natural Astragalus membranaceus. At the same time, the target protein was detected to be with nuclease activity, which provide a basis for the study of related activities of exogenous AmPR-10.

Key words: Astragalus membranaceus, pathogenesis-related protein, soluble expression, tag, molecular chaperones