重组人瘦素蛋白的原核表达、纯化和生物学活性鉴定

doi:10.3969/j.issn.1000-1565.2022.05.010

摘要/Abstract

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

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- 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 Xiangyang^1,2, LI Zhongkai³, HAO Tengfei³, DING Guangshuo⁴, ZHENG Wenhui¹, LI Wenjuan^1,2(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.

Key words: recombinant hLeptin, SUMO, prokaryotic expression, biological activity

中图分类号:

周向阳,李仲凯,郝腾飞,丁光硕,郑文慧,李文娟. 重组人瘦素蛋白的原核表达、纯化和生物学活性鉴定[J]. 河北大学学报(自然科学版), 2022, 42(5): 511-518.

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.

参考文献

[1] REHMAN K, AKASH M S H, ALINA Z.Leptin: a new therapeutic target for treatment of diabetes mellitus[J]. J Cell Biochem, 2018, 119(7): 5016-5027. DOI:10.1002/jcb.26580.
[2] FUNCKE J B, VON SCHNURBEIN J, LENNERZ B, et al. Monogenic forms of childhood obesity due to mutations in the leptin gene[J]. Mol Cell Pediatr, 2014, 1: 3. DOI:10.1186/s40348-014-0003-1.
[3] MYERS M G Jr, LEIBEL R L, SEELEY R J, et al. Obesity and leptin resistance: distinguishing cause from effect[J]. Trends Endocrinol Metab, 2010, 21(11): 643-651. DOI:10.1016/j.tem.2010.08.002.
[4] PARK H K, AHIMA R S. Physiology of leptin: energy homeostasis, neuroendocrine function and metabolism[J]. Metabolism, 2015, 64(1): 24-34. DOI:10.1016/j.metabol.2014.08.004.
[5] RAMOS-LOBO A M, DONATO J Jr. The role of leptin in health and disease[J]. Temperature(Austin), 2017, 4(3): 258-291. DOI:10.1080/23328940.2017.1327003.
[6] KATSIKI N, MIKHAILIDIS D P, BANACH M. Leptin, cardiovascular diseases and type 2 diabetes mellitus[J]. Acta Pharmacol Sin, 2018, 39(7): 1176-1188. DOI:10.1038/aps.2018.40.
[7] GHASEMI A, SAEIDI J, AZIMI-NEJAD M, et al. Leptin-induced signaling pathways in cancer cell migration and invasion[J]. Cell Oncol, 2019, 42(3): 243-260. DOI:10.1007/s13402-019-00428-0.
[8] YACOBOVITZ M, SOLOMON G, GUSAKOVSKY E E, et al. Purification and characterization of recombinant pufferfish(takifugu rubripes)leptin[J]. Gen Comp Endocrinol, 2008, 156(1): 83-90. DOI:10.1016/j.ygcen.2007.11.013.
[9] PARK J H, LEE H H, NA S Y, et al. Recombinant expression of biologically active rat leptin in escherichia coli[J]. Protein Expr Purif, 2001, 22(1): 60-69. DOI:10.1006/prep.2001.1412.
[10] VARNERIN J P, SMITH T, ROSENBLUM C I, et al. Production of leptin inEscherichia coli: a comparison of methods[J]. Protein Expr Purif, 1998, 14(3): 335-342. DOI:10.1006/prep.1998.0978.
[11] DEMAIN A L, VAISHNAV P. Production of recombinant proteins by microbes and higher organisms[J]. Biotechnol Adv, 2009, 27(3): 297-306. DOI:10.1016/j.biotechadv.2009.01.008.
[12] YIN J C, LI G X, REN X F, et al. Select what You need: a comparative evaluation of the advantages and limitations of frequently used expression systems for foreign genes[J]. J Biotechnol, 2007, 127(3): 335-347. DOI:10.1016/j.jbiotec.2006.07.012.
[13] LOZANO TEROL G, GALLEGO-JARA J, SOLA MARTÍNEZ R A, et al. Impact of the expression system on recombinant protein production in Escherichia coli BL21[J]. Front Microbiol, 2021, 12: 682001. DOI:10.3389/fmicb.2021.682001.
[14] BÉKÉS M, PRUDDEN J, SRIKUMAR T, et al. The dynamics and mechanism of SUMO chain deconjugation by SUMO-specific proteases[J]. J Biol Chem, 2011, 286(12): 10238-10247. DOI:10.1074/jbc.M110.205153.
[15] ZUO X, MATTERN M R, TAN R, et al. Expression and purification of SARS coronavirus proteins using SUMO-fusions[J]. Protein Expr Purif, 2005, 42(1): 100-110. DOI:10.1016/j.pep.2005.02.004.
[16] MALAKHOV M P, MATTERN M R, MALAKHOVA O A, et al. SUMO fusions and SUMO-specific protease for efficient expression and purification of proteins[J]. J Struct Funct Genom, 2004, 5(1-2): 75-86. DOI:10.1023/B: JSFG.0000029237.70316.52.
[17] ZHANG Y Y, PROENCA R, MAFFEI M, et al. Positional cloning of the mouse obese gene and its human homologue[J]. Nature, 1994, 372(6505): 425-432. DOI:10.1038/372425a0.
[18] KELESIDIS T, KELESIDIS I, CHOU S, et al. Narrative review: the role of leptin in human physiology: emerging clinical applications[J]. Ann Intern Med, 2010, 152(2): 93-100. DOI:10.7326/0003-4819-152-2-201001190-00008.
[19] MÜNZBERG H, MORRISON C D. Structure, production and signaling of leptin[J]. Metabolism, 2015, 64(1): 13-23. DOI:10.1016/j.metabol.2014.09.010.
[20] CUI H X, LÓPEZ M, RAHMOUNI K. The cellular and molecular bases of leptin and ghrelin resistance in obesity[J]. Nat Rev Endocrinol, 2017, 13(6): 338-351. DOI:10.1038/nrendo.2016.222.
[21] ZHOU J, LEI W, SHEN L, et al. Primary study of leptin and human hepatocellular carcinoma in vitro[J]. World J Gastroenterol, 2008, 14(18): 2900-2904. DOI:10.3748/wjg.14.2900.
[22] STEFANOU N, PAPANIKOLAOU V, FURUKAWA Y, et al. Leptin as a critical regulator of hepatocellular carcinoma development through modulation of human telomerase reverse transcriptase[J]. BMC Cancer, 2010, 10: 442. DOI:10.1186/1471-2407-10-442.
[23] SAXENA N K, SHARMA D, DING X K, et al. Concomitant activation of the JAK/STAT, PI3K/AKT, and ERK signaling is involved in leptin-mediated promotion of invasion and migration of hepatocellular carcinoma cells[J]. Cancer Res, 2007, 67(6): 2497-2507. DOI:10.1158/0008-5472.can-06-3075.
[24] LAWLER K, HUANG-DORAN I, SONOYAMA T, et al. Leptin-mediated changes in the human metabolome[J]. J Clin Endocrinol Metab, 2020, 105(8): 2541-2552. DOI:10.1210/clinem/dgaa251.
[25] LEE J H, CHAN J L, SOURLAS E, et al. Recombinant methionyl human leptin therapy in replacement doses improves insulin resistance and metabolic profile in patients with lipoatrophy and metabolic syndrome induced by the highly active antiretroviral therapy[J]. J Clin Endocrinol Metab, 2006, 91(7): 2605-2611. DOI:10.1210/jc.2005-1545. (