title: Production of bioactive recombinant rat soluble receptor for advanced glycation end products (rrsRAGE) in Pichia pastoris.
abstract: Soluble receptor for advanced glycation end products (sRAGE), a natural inhibitor of RAGE, is considered to be a putative therapeutic molecule for a variety of diseases and a biomarker for certain conditions. To further study the function of sRAGE, recombinant rat sRAGE (rrsRAGE) was expressed and produced in a eukaryotic system. The open reading frame of rat sRAGE was cloned downstream of the methanol-inducible alcohol oxidase promoter of pPICZαA vector, and Pichia pastoris strain X-33 was used as the host strain. The expression of rrsRAGE was achieved by fermentation in a 15-L bioreactor and the resulting fermentation broth was subjected to purification on a cation exchange chromatography column. The purification of rrsRAGE reached 95% after size exclusion chromatography(SEC). The bioactivity of the purified protein was confirmed in a SH-SY5Y cell proliferation assay. The biological function of the purified rrsRAGE protein rat CCl4-induced model was then examined. Treatment with rrsRAGE resulted in significantly lower liver fibrosis and lower serum level of ALT, suggesting that sRAGE prevent liver from injury and fibrosis. In conclusion, we achieved high-efficiency production of bioactive rrsRAGE in P. pastoris.
authors.affiliation: Shanghai Municipality Key Laboratory of Veterinary Biotechnology, School of Agriculture and Biology, Shanghai Jiao Tong University, Shanghai 200240, China.
authors.forename: Peng
authors.initials: P
authors.lastname: Xia
authors.affiliation: Laboratory of Regenerative Medicine, School of Pharmacy, Shanghai Jiao Tong University, Shanghai 200240, China.
authors.forename: Jin
authors.initials: J
authors.lastname: Gao
authors.affiliation: Shanghai Municipality Key Laboratory of Veterinary Biotechnology, School of Agriculture and Biology, Shanghai Jiao Tong University, Shanghai 200240, China.
authors.forename: Wen
authors.initials: W
authors.lastname: Guan
authors.affiliation: Laboratory of Regenerative Medicine, School of Pharmacy, Shanghai Jiao Tong University, Shanghai 200240, China.
authors.forename: Jingjing
authors.initials: J
authors.lastname: Li
authors.affiliation: Shanghai Municipality Key Laboratory of Veterinary Biotechnology, School of Agriculture and Biology, Shanghai Jiao Tong University, Shanghai 200240, China.
authors.forename: Xiaolan
authors.initials: X
authors.lastname: Yu
authors.affiliation: Department of Obstetrics and Gynecology, Shanghai Jiao Tong University Affiliated First People's Hospital, Shanghai 200080, China.
authors.forename: Fangyuan
authors.initials: F
authors.lastname: Wang
authors.affiliation: Shanghai Municipality Key Laboratory of Veterinary Biotechnology, School of Agriculture and Biology, Shanghai Jiao Tong University, Shanghai 200240, China.
authors.forename: Honglin
authors.initials: H
authors.lastname: He
authors.affiliation: Shanghai Municipality Key Laboratory of Veterinary Biotechnology, School of Agriculture and Biology, Shanghai Jiao Tong University, Shanghai 200240, China.
authors.forename: Qing
authors.initials: Q
authors.lastname: Deng
authors.affiliation: Shanghai Municipality Key Laboratory of Veterinary Biotechnology, School of Agriculture and Biology, Shanghai Jiao Tong University, Shanghai 200240, China.
authors.forename: Liang
authors.initials: L
authors.lastname: Zhou
authors.affiliation: Shanghai Municipality Key Laboratory of Veterinary Biotechnology, School of Agriculture and Biology, Shanghai Jiao Tong University, Shanghai 200240, China.
authors.forename: Yunsheng
authors.initials: Y
authors.lastname: Yuan
authors.affiliation: Laboratory of Regenerative Medicine, School of Pharmacy, Shanghai Jiao Tong University, Shanghai 200240, China. Electronic address: weihan@sjtu.edu.cn.
authors.forename: Wei
authors.initials: W
authors.lastname: Han
authors.affiliation: Shanghai Municipality Key Laboratory of Veterinary Biotechnology, School of Agriculture and Biology, Shanghai Jiao Tong University, Shanghai 200240, China. Electronic address: yanyu@sjtu.edu.cn.
authors.forename: Yan
authors.initials: Y
authors.lastname: Yu
mesh_terms: D000818:Animals; D001483:Base Sequence; D019149:Bioreactors; D002251:Carbon Tetrachloride; D002252:Carbon Tetrachloride Poisoning / Q000473:pathology / Q000517:prevention & control*; D045744:Cell Line, Tumor; D002470:Cell Survival / Q000187:drug effects; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D003001:Cloning, Molecular; D005285:Fermentation; D015870:Gene Expression; D005822:Genetic Vectors / Q000737:chemistry* / Q000378:metabolism; D008103:Liver Cirrhosis / Q000139:chemically induced / Q000473:pathology / Q000517:prevention & control*; D009474:Neurons / Q000166:cytology / Q000187:drug effects; D016366:Open Reading Frames; D010843:Pichia / Q000235:genetics* / Q000378:metabolism; D011401:Promoter Regions, Genetic; D051381:Rats; D017207:Rats, Sprague-Dawley; D000067759:Receptor for Advanced Glycation End Products / Q000008:administration & dosage / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification / Q000494:pharmacology
chemical_list: C000593874:Ager protein, rat; D000067759:Receptor for Advanced Glycation End Products; D011994:Recombinant Proteins; D002251:Carbon Tetrachloride
keywords: Expression and purification; Pichia pastoris; Soluble receptor for advanced glycation end products (sRAGE)