CAS OpenIR
Expression, Purification, and Polyethylene Glycol Site-Specific Modification of Recombinant Human Interleukin 24 in Escherichia coli
Zhang, Yao1,2; Ma, Qunfeng3; Wang, Junfeng1; Ge, Jianlin1; Hua, Jilei1; Shi, Yinan1; Zhang, Chi1; Liu, Mengzhe1; Wang, Yuqi1; Chen, Zhinan4; Wang, Ziling1; Liu, Yongdong2; Jiang, Hong1
2019-10-01
Source PublicationPROTEIN JOURNAL
ISSN1572-3887
Volume38Issue:5Pages:576-585
AbstractInterleukin 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.
KeywordIL-24 Gene engineering proteins PEG site-specific modification Half-life
DOI10.1007/s10930-019-09836-5
Language英语
WOS KeywordHUMAN NONGLYCOSYLATED ERYTHROPOIETIN ; MELANOMA DIFFERENTIATION ; MDA-7 GENE ; IN-VITRO ; GROWTH ; PEGYLATION ; APOPTOSIS ; MDA-7/IL-24 ; CELLS
Funding ProjectBeijing Natural Science Foundation[7142117]
WOS Research AreaBiochemistry & Molecular Biology
WOS SubjectBiochemistry & Molecular Biology
Funding OrganizationBeijing Natural Science Foundation
WOS IDWOS:000486331800008
PublisherSPRINGER
Citation statistics
Document Type期刊论文
Identifierhttp://ir.ipe.ac.cn/handle/122111/30985
Collection中国科学院过程工程研究所
Corresponding AuthorLiu, Yongdong; Jiang, Hong
Affiliation1.Beijing Jiaotong Univ, Coll Life Sci & Bioengn, Sch Sci, 3 Shangyuancun, Beijing 100044, Peoples R China
2.Chinese Acad Sci, Natl Key Lab Biochem Engn, Inst Proc Engn, Beijing 100190, Peoples R China
3.Acad Mil Med Sci, Affiliated Hosp, Dept Thorac Surg, Beijing 100071, Peoples R China
4.Air Force Med Univ, Xian 710032, Shaanxi, Peoples R China
Recommended Citation
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.
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