title: Large-scale expression, purification, and glucose uptake activity of recombinant human FGF21 in Escherichia coli.
abstract: As a novel important regulator of glucose and lipid metabolism homeostasis, human fibroblast growth factor 21 (hFGF21) has become a potential drug candidate for the treatment of metabolic diseases including obesity, and type 2 diabetes, as well as non-alcoholic fatty liver disease. To improve the production of recombinant hFGF21 to meet the increasing demand in clinical applications, an artificial gene encoding its mature peptide sequence was constructed, cloned into vector pET-3c and then expressed in Escherichia coli Origami B (DE3). Under optimal conditions in a 50-L fermentor, the average bacterial yield and the soluble expression level of recombinant hFGF21 of six batches attained 1750 ± 185 g and 32 ± 1.5%, respectively. The target protein was purified by the combination of nickel-nitrilotriacetic acid affinity chromatography and Sephadex S-100 resin. 5% (w/v) trehalose solution was able to prevent rhFGF21 from degradation effectively. The purity of rhFGF21 was higher than 97%, and the yield was 213 ± 17 mg/L. The preliminary biochemical characterization of rhFGF21 was confirmed using Western blot and peptide map finger analysis. Based on the glucose oxidase-peroxidase assay, the EC50 of glucose uptake activity of the purified rhFGF21 was 22.1 nM.
authors.affiliation: Institute of Life and Health Engineering, and National Engineering Research Center of Genetic Medicine, Jinan University, Guangzhou, 510632, People's Republic of China.
authors.forename: Minjing
authors.initials: M
authors.lastname: Zhang
authors.forename: Xuebin
authors.initials: X
authors.lastname: Jiang
authors.forename: Zhijian
authors.initials: Z
authors.lastname: Su
authors.forename: Jiancong
authors.initials: J
authors.lastname: Lin
authors.forename: Qi
authors.initials: Q
authors.lastname: Xiang
authors.forename: Zhan
authors.initials: Z
authors.lastname: Yang
authors.forename: Yadong
authors.initials: Y
authors.lastname: Huang
authors.forename: Xiaokun
authors.initials: X
authors.lastname: Li
mesh_terms: D019149:Bioreactors; D001709:Biotechnology / Q000379:methods; D002853:Chromatography, Liquid / Q000379:methods; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005346:Fibroblast Growth Factors / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D015870:Gene Expression; D005813:Genes, Synthetic; D005822:Genetic Vectors; D005947:Glucose / Q000378:metabolism*; D006801:Humans; D010957:Plasmids; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism
chemical_list: D011994:Recombinant Proteins; C414620:fibroblast growth factor 21; D005346:Fibroblast Growth Factors; D005947:Glucose