重组野生型和酶活缺失型人ENO1蛋白在昆虫细胞/杆状病毒表达系统内的表达、纯化及酶活测定

Expression, purification, and enzyme activity determination of recombinant wild-type and active site-deleted human ENO1 proteins in insect cell/baculovirus expression system

摘要:

目的 利用昆虫细胞/杆状病毒表达系统高效表达并纯化野生型及3种酶活位点缺失型(ENO1-M1、ENO1-M2、ENO1-M3)重组人烯醇化酶1 (ENO1)蛋白，为后续功能研究、抗体开发及抑制剂筛选提供重要的理论基础。方法 通过分子克隆技术将C端融合6×His标签的野生型及酶活缺失型ENO1基因构建至pFastBac-HTB载体，并转化至DH10Bac感受态细胞，经筛选获得重组杆粒(bacmid)；将重组杆粒转染ExpiSf9昆虫细胞，获得P0代病毒，经扩增获得高滴度P1代病毒用于感染细胞表达目的蛋白；通过亲和层析(如镍柱)纯化表达产物，并利用SDS-PAGE、考马斯亮蓝染色及Western blotting进行鉴定；使用抗ENO1单克隆抗体、单链抗体及嵌合抗体进行Western blotting特异性检测；基于ENO1催化2-磷酸甘油酸生成磷酸烯醇式丙酮酸(phosphoenolpyruvate, PEP)的反应，通过紫外分光光度法在240 nm处测定吸光度变化，计算酶比活力。结果 成功构建了所有重组质粒与杆粒，并在昆虫细胞中实现了重组蛋白的高效表达。纯化获得了高纯度的重组蛋白，Western blotting证实其均具有反应原性，且酶活位点缺失不影响其免疫反应性。酶活测定表明，野生型ENO1的比活力为610.14 U/mg，而3种酶活缺失型蛋白的酶活显著降低，其中缺失GSHAGNK (残基156-162)和SPDDPSRYI (残基262-270)位点的ENO1-M2和ENO1-M3蛋白活性丧失尤为明显。结论 本研究成功利用昆虫细胞/杆状病毒表达系统(baculovirus expression vector system, BEVS)表达并纯化了具有反应原性的野生型及酶活缺失型ENO1蛋白，证实了GSHAGNK和SPDDPSRYI是2个关键的酶活性位点，为深入探究ENO1功能及开发其特异性抑制剂奠定了重要的理论与材料基础。

Abstract:

Objective To efficiently express and purify wild-type and three active site-deleted variants (ENO1-M1, ENO1-M2, and ENO1-M3) of ENO1 through the insect cell/baculovirus expression system, thus providing a theoretical basis for subsequent functional research, antibody development, and inhibitor screening.Methods Wild-type and active site-deleted genes of ENO1 with a C-terminal 6×His tag were cloned into the pFastBac-HTB vector via molecular cloning and then transformed into DH10Bac competent cells to obtain recombinant Bacmid through screening. Recombinant baculovirus particles were transfected into ExpiSf9 insect cells to generate the P0 virus stock, which was then amplified to produce high-titer P1 virus for target protein expression. Expression products were purified by affinity chromatography (e.g., nickel column) and identified by SDS-PAGE, Coomassie Brilliant Blue staining, and Western blotting. The anti-ENO1 monoclonal antibody, single-chain antibody, and chimeric antibody were used for specific detection by Western blotting. On the basis of the conversion of 2-phosphoglycerate to phosphoenolpyruvate (PEP) under the catalysis by ENO1, the specific activity of ENO1 was measured by UV spectrophotometry at 240 nm.Results All recombinant plasmids and bacmids were successfully constructed, and high-level expression of recombinant proteins was achieved in insect cells. High-purity recombinant proteins were obtained, and Western blotting analysis confirmed their reactivity. The deletion of active sites of the enzyme did not affect the immunoreactivity. The specific activity of wild-type ENO1 was 691.28 U/mg, while those of the three deletion variants were significantly reduced. ENO1-M2 (with deletion of GSHAGNK at residues 156-162) and ENO1-M3 (with deletion of SPDPSRYI at residues 262-270) exhibited particularly pronounced losses of enzyme activity.Conclusion We successfully expressed and purified both wild-type and active site-deleted ENO1 proteins with retained reactivity by using the baculovirus expression vector system. The results confirm that GSHAGNK and SPDPSRYI are two key active sites of this enzyme, laying both theoretical and material foundations for further exploring the functions of ENO1 and developing its specific inhibitors.