Expression, Purification, and Polyethylene Glycol Site-Specific Modification of Recombinant Human Interleukin 24 in Escherichia coli

doi:10.1007/s10930-019-09836-5

	Expression, Purification, and Polyethylene Glycol Site-Specific Modification of Recombinant Human Interleukin 24 in Escherichia coli
	Zhang, Yao 1,4; Ma, Qunfeng 3; Wang, Junfeng 1; Ge, Jianlin 1; Hua, Jilei 1; Shi, Yinan 1; Zhang, Chi 1; Liu, Mengzhe 1; Wang, Yuqi 1; Chen, Zhinan 2
刊名	PROTEIN JOURNAL
	2019-10-01
卷号	38 期号:5 页码:576-585
关键词	IL-24 Gene engineering proteins PEG site-specific modification Half-life
ISSN号	1572-3887
DOI	10.1007/s10930-019-09836-5
英文摘要	Interleukin 24 (IL-24) has a broad spectrum of specific antitumor activities without affecting normal cells. The recombinant human IL-24 (rhIL-24) expressed in E. coli has low biological activity due to lack of necessary glycosylation modification. In this study, based on the modification of the non-glycosylated IL-24 with polyethylene glycol (PEG), we aimed to improve the stability and prolong its half-life in vivo. Firstly, the recombinant plasmid containing the hIL-24 cDNA was prepared by the prokaryotic-expression plasmid pET-28a and transformed into E. coli BL21. After induced by isopropyl beta-D-thiogalactoside (IPTG), the target protein rhIL-24 was expressed as insoluble inclusion body, which was solubilized and denatured by 6 M guanidine hydrochloride. The denatured rhIL-24 was diluted to refold in the optimized buffer overnight at the protein concentration of 0.1 mg/mL. The refolded rhIL-24 was mainly in the form of soluble aggregate, but high-purity monomer rhIL-24 was obtained through size exchange chromatography with the addition of SDS in elution buffer. The tertiary structure of rhIL-24 was confirmed by fluorescence spectroscopy. Western blot analysis showed that rhIL-24 could be site-specifically modified by mPEG5000-ALD. Methyl thiazolyl tetrazolium (MTT) assay showed no significant difference between mPEG5000-ALD-rhIL-24 and rhIL-24 in inhibiting the growth of melanoma cell line A375 in vitro. Pharmacokinetic studies showed that PEG modification could significantly improve the stability and prolong the half-life of rhIL-24 from 8.41 to 13.2 h. The data strongly suggested that mPEG-ALD 5000 could site-specifically modify rhIL-24 expressed in E. coli. The PEG modification significantly prolonged the half-life of rhIL-24 without reducing its antitumor activity in vitro.
资助项目	Beijing Natural Science Foundation[7142117]
WOS关键词	HUMAN NONGLYCOSYLATED ERYTHROPOIETIN ; MELANOMA DIFFERENTIATION ; MDA-7 GENE ; IN-VITRO ; GROWTH ; PEGYLATION ; APOPTOSIS ; MDA-7/IL-24 ; CELLS
WOS研究方向	Biochemistry & Molecular Biology
语种	英语
出版者	SPRINGER
WOS记录号	WOS:000486331800008
资助机构	Beijing Natural Science Foundation
内容类型	期刊论文
源URL	[http://ir.ipe.ac.cn/handle/122111/30984]
专题	中国科学院过程工程研究所
通讯作者	Liu, Yongdong; Jiang, Hong
作者单位	1.Beijing Jiaotong Univ, Coll Life Sci & Bioengn, Sch Sci, 3 Shangyuancun, Beijing 100044, Peoples R China 2.Air Force Med Univ, Xian 710032, Shaanxi, Peoples R China 3.Acad Mil Med Sci, Affiliated Hosp, Dept Thorac Surg, Beijing 100071, Peoples R China 4.Chinese Acad Sci, Natl Key Lab Biochem Engn, Inst Proc Engn, Beijing 100190, Peoples R China
推荐引用方式 GB/T 7714	Zhang, Yao,Ma, Qunfeng,Wang, Junfeng,et al. Expression, Purification, and Polyethylene Glycol Site-Specific Modification of Recombinant Human Interleukin 24 in Escherichia coli[J]. PROTEIN JOURNAL,2019,38(5):576-585.
APA	Zhang, Yao.,Ma, Qunfeng.,Wang, Junfeng.,Ge, Jianlin.,Hua, Jilei.,...&Jiang, Hong.(2019).Expression, Purification, and Polyethylene Glycol Site-Specific Modification of Recombinant Human Interleukin 24 in Escherichia coli.PROTEIN JOURNAL,38(5),576-585.
MLA	Zhang, Yao,et al."Expression, Purification, and Polyethylene Glycol Site-Specific Modification of Recombinant Human Interleukin 24 in Escherichia coli".PROTEIN JOURNAL 38.5(2019):576-585.