小泛蛋白样修饰物双荧光融合蛋白及其特异性蛋白酶的原核表达及鉴定

Prokaryotic expression and identification of dual-fluorescence fusion proteins of small ubiquitin-like modifier and sentrin-specific protease

摘要:

本实验克隆了人的4种SENP(Sentrin-specific protease)C端催化结构域、3种SUMO(Small ubiquitin-like modifer)、ECFP(Enhanced cyan fluorescent protein)以及EYFP(Enhanced yellow fluorescent protein)的基因, 通过RecJoin克隆技术分别成功构建SENP和ECFP-SUMO-EYFP表达载体B28和B13。将表达载体转化大肠杆菌BL21, 经IPTG诱导表达, 通过Ni-NTA离子交换柱层析纯化, 进一步采用SDS-PAGE和Western blotting分析鉴定, 并以酶切实验初步分析了SENP相对于底物ECFP-SUMO-EYFP的酶切特异性。最终, SENP3C(SENP3 catalytic domain)截短表达, SENP5C以包涵体形式表达, 其他蛋白均为可溶性表达, SDS-PAGE分析表明表达产物分子质量(Mr)与预期相符, Western blotting方法进一步证实其为目的蛋白。酶切实验初步鉴定了SENP1C和SENP2C的酶切特异性。以上蛋白的原核表达为荧光共振能量转移实验奠定了实验基础。

Abstract:

We cloned genes of four sentrin-specific protease (SENP), three small ubiquitin-like modifiers (SUMO), enhanced cyan fluorescent protein (ECFP) and yellow fluorescent protein (EYFP) by two-step PCR. Then we constructed expression vector B28 for SENP and B13 for ECFP-SUMO-EYFP. The recombinant plasmids were transformed into Escherichia coli BL21 and expression was induced by isopropyl β-D-thiogalactoside, then recombinant proteins were purified by Ni-NTA agarose column ion-exchange chromatography. The proteins were analyzed with SDS-PAGE and identified with Western blotting. Except that SENP3 catalytic domain (SENP3C) truncated in the C termini and SENP5C expressed in inclusion body, others were expressed as soluble proteins. SDS-PAGE analysis showed that the relative molecular mass of these fusion proteins were consistent with theoretical ones, and the specificity of the fusion proteins were confirmed with Western blotting. The fusion proteins of the SENP and ECFP-SUMO-EYFP can be successfully expressed in prokaryotic expression system. It lays the foundation for the fluorescence resonance energy transfer analysis.