To avoid the formation of inclusion bodies and the low yield caused by protein aggregation in the process of refolding in vitro, during the large-scale production of recombinant human leptin protein(hLeptin), the high-level soluble expression of hLeptin in Escherichia coli was investigated, and the biological activity was detected. The fusion expression plasmid pET-30a(+)-SUMO-Lep was constructed by using the coding sequence of mature peptide of hLeptin and pET-30a(+)-SUMO system, which was transformed into E.coli BL21(DE3)and was induced by isopropyl-β-D-thiogalactose(IPTG). Afterwards, the recombinant protein was purified by Ni metal affinity chromatography, and the mature hLeptin- DOI:10.3969/j.issn.1000-1565.2022.05.010重组人瘦素蛋白的原核表达、纯化和生物学活性鉴定周向阳1,2,李仲凯3,郝腾飞3,丁光硕4,郑文慧1,李文娟1,2(1.河北大学 基础医学院,河北 保定 071000;2.河北省炎性自身免疫性疾病发病机制及防治重点实验室,河北 保定 071000;3.河北农业大学 生命科学学院,河北 保定 071001;4.河北大学 公共卫生学院,河北 保定 071000)摘 要:为解决重组人瘦素蛋白(hLeptin)大规模生产过程中包涵体的形成以及体外重折叠过程中蛋白聚集导致的低产量,探究在大肠杆菌中hLeptin高水平可溶性的表达,并进行生物学活性检测.利用hLeptin成熟肽基因与pET-30a(+)-SUMO系统构建融合表达质粒pET-30a(+)-SUMO-Lep,转化至大肠杆菌BL21(DE3),加入异丙基-β-D-硫代半乳糖(IPTG)进行诱导表达,之后经Ni金属亲和层析纯化,并切去SUMO融合标签后获得成熟hLeptin.重组hLeptin表达量高,主要以可溶形式存在.通过KM小鼠减肥实验和CCK8实验证明重组hLeptin具有生物学活性.综上,使用SUMO融合标签,以增强hLeptin在大肠杆菌中的高效可溶性高纯度的表达,为大规模生产hLeptin提供了一种新方法,同时为表达可溶性重组蛋白研究提供了一定的基础.关键词:重组hLeptin;SUMO;原核表达;生物学活性中图分类号:Q78 文献标志码:A 文章编号:1000-1565(2022)05-0511-08Prokaryotic expression,purification and biological activity identification of recombinant human leptin proteinZHOU Xiangyang1,2, LI Zhongkai3, HAO Tengfei3, DING Guangshuo4, ZHENG Wenhui1, LI Wenjuan1,2(1.School of Basic Medicine Sciences, Hebei University, Baoding 071000, China;2.Key Laboratory of Pathogenesis Mechanism and Control of Inflammatory Autoimmune Diseases in Hebei Province, Baoding 071000, China;3.College of Life Sciences, Hebei Agricultural University, Baoding 071001, China;4.College of Public Health, Hebei University, Baoding 071000, China)Abstract: To avoid the formation of inclusion bodies and the low yield caused by protein aggregation in the process of refolding in vitro, during the large-scale production of recombinant human leptin protein(hLeptin), the high-level soluble expression of hLeptin in Escherichia coli was investigated, and the biological activity was detected. The fusion expression plasmid pET-30a(+)-SUMO-Lep was constructed by using the coding sequence of mature peptide of hLeptin and pET-30a(+)-SUMO system, which was transformed into E.coli BL21(DE3)and was induced by isopropyl-β-D-thiogalactose(IPTG). Afterwards, the recombinant protein was purified by Ni metal affinity chromatography, and the mature hLeptin- 收稿日期:2022-03-02 基金项目:河北省自然科学基金资助项目(H2019201084);河北大学医学学科培育项目(2021A01);河北大学大学生创新创业项目(2022309) 第一作者:周向阳(1997—),男,山西大同人,河北大学在读硕士研究生,主要从事肿瘤代谢及化学预防的研究.E-mail: zxy9708@163.com 通信作者:李文娟(1977—),女,甘肃静宁人,河北大学副教授,博士,主要从事肿瘤代谢研究.E-mail: liwenjuan@hbu.edu.cn第5期周向阳等:重组人瘦素蛋白的原核表达、纯化和生物学活性鉴定was obtained after cutting off the SUMO fusion tag. The recombinant hLeptin was highly expressed and mainly existed in soluble form. The biological activity was confirmed by KM mouse Weight-Losing Test and the CCK8 assay. In this study, we employed the SUMO fusion tag to increase the solubility and the purity of hLeptin expression in E. coli, which could provide a novel method for large-scale production of leptin and lay a foundation for the expression of soluble recombinant protein.
ZHOU Xiangyang
,
LI Zhongkai
,
HAO Tengfei
,
DING Guangshuo
,
ZHENG Wenhui
,
LI Wenjuan
. Prokaryotic expression,purification and biological activity identification of recombinant human leptin protein[J]. Journal of Hebei University(Natural Science Edition), 2022
, 42(5)
: 511
-518
.
DOI: 10.3969/j.issn.1000-1565.2022.05.010
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