人蛋白酶体亚基PSMB1的重组表达、纯化及在蛋白酶体抑制剂体外筛选中的应用

Expression, purification of poteasome subunit PSMB1 and application in screening of possible proteasome inhibitors

摘要:

蛋白酶体是真核细胞中的一类多亚基蛋白酶复合物，它在胞内蛋白质降解的泛素-蛋白酶体通路中起关键作用。重组表达蛋白酶体的活性亚基可以用于在体外筛选、寻找具有蛋白酶体抑制剂作用的化合物。将人蛋白酶体催化亚基（PSMB1）cDNA的编码区（全长726 bp）克隆至原核表达载体pET28a(+)，构建重组质粒pET28a-PSMB1，转化大肠杆菌BL21(DE3)，通过1 mmol/L IPTG，20 ℃过夜诱导，获得相对分子量约为27 kDa的重组蛋白，采用IMAC亲和层析柱纯化重组蛋白，纯化后的重组蛋白纯度超过95％。重组蛋白酶解后经NanoLC-MS/MS鉴定表明所表达的融合蛋白氨基酸序列完全正确。在体外BIAcore分析中，重组蛋白表现出对不同化合物的选择性结合能力，其中与蛋白酶体抑制剂雷公藤红素的结合较强，10 μmol/L的雷公藤红素与重组蛋白的结合达到27 RU，并且具有良好的浓度依赖型。本研究建立了表达、纯化人蛋白酶体催化亚基PSMB1的方法，并应用于具有蛋白酶体抑制活性化合物的体外筛选。

Abstract:

Proteasome is a multi-subunit protease complex in eukaryocytes, and plays an important role in ubiquitin-proteosome pathway. Recombinant proteasome can be used to screen proteasome inhibitors. In this study, recombinant plasmid of pET28a-PSMB1 was constructed by inserting human proteasome catalytic subunit (PSMB1) cDNA (726bp) into the prokaryotic expression vector pET28a(+), and transforming the plasmid into E. coli BL21(DE3) cells for expression. After overnight induction (1 mmol/L IPTG, 20 °C), an expected protein band with molecular weight of 27 kDa was observed on SDS-PAGE gel. The recombinant protein was then purified through affinity chromatography, and the purity is more than 95%. The amino acid sequence of the recombinant protein was validated by NanoLC-MS/MS. The data from in vitro BIAcore analysis showed that the recombinant PSMB1 could bind to celastrol. The binding affinity between PSMB1 and 10 μmol/L celastrol was more than 27RU.