Large-scale expression, purification, and glucose uptake activity of recombinant human FGF21 in Escherichia coli
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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.
KeywordsHuman fibroblast growth factor 21 Expression Purification Glucose uptake activity
This work was supported by grants of the National Natural Science Foundation of China (No.31000663), National Key New Drug Foundation of China (No.2011ZX09102-004-03 and 2009ZX09103-749), the Science Foundation of Wenzhou of China (No.Y20090279), the Science Foundation of Guangdong of China (No. 2009B080701090 and 2007B030700004), and the Program for New Century Excellent Talents in University (NCET-08-0611).
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