(L)-2-羟基戊二酸脱氢酶的原核表达与活性鉴定  

(L)-2-hydroxyglutarate Dehydrogenase Expression in Prokaryotes and Determination of Enzyme Activity

作　　者：李俊辉 王璐 谷雨 叶菁 袁媛 LI Jun-hui;WANG Lu;GU Yu;YE Jing;YUAN Yuan(Department of Pathology,Basic Medical Science Academy,Air Force Medical University,Xi'an,Shaanxi,710032,China;Department of Otolaryngology,the Second Affiliated Hospital of Air Force Military Medical University,Xi'an,Shaanxi,710038,China)

机构地区：[1]空军军医大学基础医学院病理学教研室,陕西西安710032 [2]空军军医大学第二附属医院耳鼻喉科,陕西西安710038

出　　处：《现代生物医学进展》2025年第1期19-25,共7页Progress in Modern Biomedicine

基　　金：国家自然科学基金项目(82170589);陕西省重点产业创新链重点项目(2019ZDLSF02-01);西京医院助推课题(XJZT24CY01)。

摘　　要：目的:构建带有6×His标签的大肠杆菌(L)-2-羟基戊二酸脱氢酶(L2HGDH)的原核表达载体,并进行重组蛋白的原核表达和纯化,以及酶活性鉴定。方法:以GenBank中大肠埃希菌K12菌株(Escherichia coli strain K12)的L2HGDH编码基因为模板,将其克隆到pET-28a(+)载体质粒中,在BL21(DE3)菌中进行诱导表达,进一步采用Ni-NTA系统纯化L2HGDH蛋白,利用SDS-PAGE及Western印迹检测蛋白纯化效果。纯化的L2HGDH催化(L)-2-羟基戊二酸(L-2HG)生成α-酮戊二酸(α-KG),时还原烟酰胺腺嘌呤二核苷酸(NAD+)为NADH,利用NADH在340 nm处有最大吸光度值,分析获得的大肠杆菌L2HGDH的酶活性,以及温度和pH值等对其酶活性的影响。结果:成功构建pET-28a(+)-L2HGDH原核表达质粒,并在BL21(DE3)中获得可溶性重组蛋白,获得L2HGDH蛋白纯度大于90%,且具有较高的(L)-2-羟基戊二酸脱氢酶生物学活性。结论:成功原核表达并纯化L2HGDH重组蛋白,重组蛋白具有良好的生物学活性,为研究L2HGDH的功能奠定了基础。Objective:To construct a prokaryotic expression vector for Escherichia coli(L)-2-hydroxyglutarate dehydrogenase(L2HGDH)incorporating a 6×His tag.Subsequently,the recombinant protein will be expressed and purified in E.coli,followed by the determination of the enzyme activity.Methods:The L2HGDH gene of the Escherichia coli K12 strain in GenBank was cloned into the pET-28a(+)vector plasmid and induced expression in BL21(DE3).The L2HGDH protein was purified by using the Ni-NTA system.SDS-PAGE and Western blot were employed to assess the efficiency of the protein purification.Purified L2HGDH catalyses the conversion of(L)-2-hydroxyglutaric acid(L-2HG)toα-ketoglutaric acid(α-KG)and reduces nicotinamide adenine dinucleotide(NAD+)to NADH.The enzymatic activity of the L2HGDH obtained from E.coli was analyzed by using the maximum absorbance value at 340 nm,and the effects of temperature and pH value on its enzymatic activity were determined.Results:The prokaryotic expression plasmid pET-28a(+)-L2HGDH was successfully constructed,and the soluble recombinant protein was obtained in BL21(DE3).The purity of the L2HGDH protein was greater than 90%,and the L2HGDH protein exhibited a high biological activity of(L)-2-hydroxyglutarate dehydrogenase.Conclusions:The recombinant L2HGDH protein was successfully expressed and purified in Escherichia coli,and the recombinant protein exhibited biological activity.This provided a foundation for further studies on the function of L2HGDH.

关 键 词：(L)-2-羟基戊二酸脱氢酶 原核表达 蛋白纯化 酶活性 

分 类 号：R3[医药卫生—基础医学] R34[生物学—生物化学] Q554