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An efficient large-scale refolding technique for recovering biologically active recombinant human FGF-21 from inclusion bodies

Ye, Xianlong, Yu, Dan, Wu, Yunzhou, Han, Jun, Li, Shujie, Wu, Qiang, Li, Deshan, Qi, Jianying
International journal of biological macromolecules 2019 v.135 pp. 362-372
Escherichia coli, affinity chromatography, bacteria, diabetes, disulfide bonds, drugs, fermentation, fibroblast growth factors, glucose, glycemic effect, glycolipids, human cell lines, humans, inclusion bodies, mice, microfiltration, molecular weight, obesity, proteins
Fibroblast growth factor 21 (FGF-21) is an important regulator in glycolipid metabolism that is a promising drug candidate for treatment of diabetes and obesity. However, the productivity of recombinant hFGF-21 (rhFGF-21) in Escherichia coli (E. coli) is relatively low, which limits its clinical application. To meet the clinical demand and control the production cost, rhFGF-21 proteins were expressed in inclusion bodies (IBs) form in Rosetta (DE3) by high cell density fermentation in 50-L scale. Hollow fiber membrane filtration technology was used to enrich the bacteria, wash, denature and refold the IBs in the current report. The renatured proteins were purified by two-step affinity chromatography. Authenticity of the purified rhFGF-21 was confirmed by the N-and C-terminal sequence, disulfide bond composition and molecular weight analyses. Results showed that the average target protein and recovery of rhFGF-21 expressed in IBs form of three batches were more than those of the soluble form. Both the rhFGF-21 proteins from the two forms showed equal potency in improving the glucose uptake in HepG2 cells and anti-diabetic effect in db/db mice. In this study, an efficient method for preparation of FGF-21 was established. This novel process provides an important technical basis for the large-scale production of rhFGF-21.