SUMO蛋白酶的发酵和纯化工艺的研究

目前, 小分子肽多需要进行融合表达，虽然有GST标签等表达体系，但是表达产物切割时仍留有多余氨基酸，影响小分子肽的功能；SUMO蛋白酶对SUMO融合表达系统表达的重组蛋白进行切割时没有多余氨基酸残留，因此成为蛋白切割工具的热点。利用基因工程技术构建重组His-Ulp1/pET3c/BL21（DE3）工程菌株，用摇瓶优化表达条件，摸索高密度发酵工艺和不同层析纯化工艺条件。结果表明，经1.0mmol/L的IPTG 30℃诱导表达6h，表达效果最好。罐发酵后菌体SDSPAGE分析表达量可达24.39%，通过CM Sepharose Fast Flow阳离子交换一步层析可获得纯度大于98%的SUMO蛋白酶，每升发酵液可获得355mg的SUMO蛋白酶纯品。Western blot分析表明，UlP1能与6×His抗体产生免疫反应。为日后大规模产业化生产奠定了基础。

Processing Technology Research of Fermentation and Purification of SUMO Protease UlP1

Abstract Nowadays, small peptides are always expressed in the form of fusion protein. The expression product contains many superfluous amino acids which can affect the biological functions of small peptides even expressed by GST fusion protein expression system. SUMO protease can cut SUMO fusion protein expressed by fusion expression system without any amino acid residues left on target protein thus become a hot topic in this field. In this study, recombinant His-UlP1/pET3c/BL21（DE3）engineering strain was constructed by genetic engineering technology and the expression conditions were optimized in shake flaks. The process of high density fermentation was explored and different purification conditions were detected by chromatography. The results showed that SUMO protease could be expressed well after inducing the engineering strain by IPTG of 1.0mmol/L at 30℃ for 6 hours. The expression level of the strain in fermentation pots could reach 24.3% analyzed by SDS-PAGE. The purity of SUMO protease was more than 98% after further purification by cation exchange chromatography. The yield was 355mg SUMO protease per liter fermentation liquid. Western blot analysis demonstrated that there were immune reactions between IlP1 and 6xHis antibodies. The study has established a good foundation for large-scale industralazation in the future.