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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,10μmol/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。
The proteasome is a multi-subunit protease complex in eukaryotic cells that plays a key role in the ubiquitin-proteasomal pathway of intracellular protein degradation. Recombinant expression of the proteasome active subunit can be used in vitro screening, looking for compounds with proteasome inhibitors. The coding region (726 bp in length) of human proteasome catalytic subunit (PSMB1) cDNA was cloned into prokaryotic expression vector pET28a (+). The recombinant plasmid pET28a-PSMB1 was transformed into E. coli BL21 (DE3) IPTG and induced overnight at 20 ℃. The recombinant protein with relative molecular weight of about 27 kDa was obtained. The recombinant protein was purified by IMAC affinity chromatography and the purity of purified recombinant protein was more than 95%. The recombinant protein was identified by NanoLC-MS / MS after proteolysis, indicating that the expressed fusion protein had the correct amino acid sequence. In in vitro BIAcore analysis, the recombinant proteins showed selective binding ability to different compounds, of which the binding to the proteasome inhibitor tripterine was stronger and the binding of 10μmol / L tripterine to the recombinant protein reached 27 RU , And has a good concentration-dependent. In this study, a method for expression and purification of human proteasome catalytic subunit PSMB1 was established and applied to in vitro screening of compounds with proteasome inhibitory activity.