title: Sortase A-aided Escherichia coli expression system for functional osteoprotegerin cysteine-rich domain.
abstract: As a natural inhibitor of the receptor activator of nuclear factor-кB ligand (RANKL), osteprotegerin (OPG) is considered a promising treatment for metabolic bone diseases. Typical approaches for preparing recombinant OPG or its derivatives employ eukaryotic expression systems. Due to the advantages of a prokaryotic expression system, which include its convenience, low cost, and abundant production, in this study, we establish a strategy for preparing functional OPG using the Escherichia coli expression system. After initial failures in preparation of OPG and its truncation, OPG cysteine-rich domain (OPG-CRD/OPGT) by using pET and pGEX vectors, we constructed a sortase A (SrtA)-aided E. coli expression system, in which the expressed protein was a self-cleaving SrtA fusion protein. Using this system, we successfully prepared the recombinant OPGT protein. The BIAcore analyses indicated that the prepared OPGT had high affinities in binding with RANKL and TRAIL. Cell experiments confirmed the inhibitory effects of the prepared OPGT on RANKL-induced osteoclast differentiation and TRAIL-induced tumor cell apoptosis. The sortase A-aided E. coli expression system for OPGT established in this study may contribute to further studies and commercial applications of OPG.
authors.affiliation: The People's Liberation Army General Hospital, 28 Fuxing road, Beijing, 100853, China.
authors.forename: Mengmeng
authors.initials: M
authors.lastname: Jin
authors.affiliation: CAS Key Laboratory of Pathogenic Microbiology and Immunology, Institute of Microbiology, Chinese Academy of Sciences, 1 Beichen Xilu, Beijing, 100101, China.
authors.forename: Yuan
authors.initials: Y
authors.lastname: Chen
authors.affiliation: CAS Key Laboratory of Pathogenic Microbiology and Immunology, Institute of Microbiology, Chinese Academy of Sciences, 1 Beichen Xilu, Beijing, 100101, China.
authors.forename: Yunfeng
authors.initials: Y
authors.lastname: Zhao
authors.affiliation: The People's Liberation Army General Hospital, 28 Fuxing road, Beijing, 100853, China.
authors.forename: Luyang
authors.initials: L
authors.lastname: Che
authors.affiliation: Beijing Key Laboratory of Research of Chinese Medicine on Prevention and Treatment for Major Diseases, Experimental Research Center, China Academy of Chinese Medical Sciences, NO.16, Dongzhimennei South Street, Dongcheng District, Beijing, 100700, China.
authors.forename: Yanyan
authors.initials: Y
authors.lastname: Ma
authors.affiliation: Beijing Key Laboratory of Research of Chinese Medicine on Prevention and Treatment for Major Diseases, Experimental Research Center, China Academy of Chinese Medical Sciences, NO.16, Dongzhimennei South Street, Dongcheng District, Beijing, 100700, China.
authors.forename: Jingzhe
authors.initials: J
authors.lastname: Li
authors.affiliation: Beijing Key Laboratory of Research of Chinese Medicine on Prevention and Treatment for Major Diseases, Experimental Research Center, China Academy of Chinese Medical Sciences, NO.16, Dongzhimennei South Street, Dongcheng District, Beijing, 100700, China.
authors.forename: Yi
authors.initials: Y
authors.lastname: Wang
authors.affiliation: CAS Key Laboratory of Pathogenic Microbiology and Immunology, Institute of Microbiology, Chinese Academy of Sciences, 1 Beichen Xilu, Beijing, 100101, China.
authors.forename: Hua
authors.initials: H
authors.lastname: Tao
authors.affiliation: CAS Key Laboratory of Pathogenic Microbiology and Immunology, Institute of Microbiology, Chinese Academy of Sciences, 1 Beichen Xilu, Beijing, 100101, China.
authors.forename: Juan
authors.initials: J
authors.lastname: Ma
authors.affiliation: Beijing Jiquan Biology Technology Co., LTD, No.8 HaiYing Rd., Fengtai District, Beijing, 100070, China.
authors.forename: Bing
authors.initials: B
authors.lastname: Pan
authors.affiliation: Beijing Key Laboratory of Research of Chinese Medicine on Prevention and Treatment for Major Diseases, Experimental Research Center, China Academy of Chinese Medical Sciences, NO.16, Dongzhimennei South Street, Dongcheng District, Beijing, 100700, China. lcz0220@163.com.
authors.forename: Changzhen
authors.initials: C
authors.lastname: Liu
authors.affiliation: The People's Liberation Army General Hospital, 28 Fuxing road, Beijing, 100853, China. harryhp@vip.sina.com.
authors.forename: Peng
authors.initials: P
authors.lastname: Huang
mesh_terms: D019881:Aminoacyltransferases / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D017209:Apoptosis / Q000187:drug effects; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D002454:Cell Differentiation / Q000187:drug effects; D003545:Cysteine / Q000737:chemistry* / Q000235:genetics; D003546:Cysteine Endopeptidases / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics*; D005822:Genetic Vectors; D006801:Humans; D051379:Mice; D010010:Osteoclasts / Q000187:drug effects; D053244:Osteoprotegerin / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics* / Q000494:pharmacology; D011485:Protein Binding; D000072417:Protein Domains; D053245:RANK Ligand / Q000494:pharmacology; D000067996:RAW 264.7 Cells; D011994:Recombinant Proteins / Q000378:metabolism; D053221:TNF-Related Apoptosis-Inducing Ligand / Q000378:metabolism / Q000494:pharmacology
chemical_list: D001426:Bacterial Proteins; D053244:Osteoprotegerin; D053245:RANK Ligand; D011994:Recombinant Proteins; D053221:TNF-Related Apoptosis-Inducing Ligand; D019881:Aminoacyltransferases; C120542:sortase A; D003546:Cysteine Endopeptidases; D003545:Cysteine
keywords: OPG-CRD/OPGT; RANKL; SrtA-aided E. coli expression system; TRAIL