Expression and purification of FGF21 in Pichia pastoris and its effect on fibroblast-cell migration

Abstract

Abstract. Fibroblast growth factor (FGF)21 functions in the maintenance of glucose homeostasis and exerts protective effects on the liver, heat and kidneys. However, the roles of FGF21 in other tissue types are yet to be fully elucidated. The present study detected elevated expression levels of FGF21 in skin tissue. Furthermore, it was revealed that FGF21 expression in the skin was induced upon wounding. In addition, β-klotho expression was detected in the skin tissue. To examine the role of FGF21 in the wound healing process, recombinant human (h)FGF21 was expressed in a the yeast strain Pichia (P.) pastoris, a well-known system for recombinant protein production. Based on the sequence of hFGF21 and the optimal codon of P. pastoris, codon-optimized FGF21 open reading frame sequences were obtained using seven pairs of 55-59-nt primers with seven rounds of PCR. The recombinant FGF21 was purified and its function was examined in human fibroblast cells using a wound healing cell migration assay. Treatment with FGF21 promoted cell migration, which is an important step in wound healing. Furthermore, FGF21 treatment enhanced the activity of c-Jun N-terminal kinase, a key regulator in fibroblast-cell migration. In conclusion, FGF21 is induced after wounding and FGF21 expressed and purified from yeast markedly accelerates wound healing. The present study was the first to elucidate the function of FGF21 in skin tissues and provided a theoretical basis for the use of FGF21 in the treatment of skin wounds. IntroductionThe mammalian genome contains 23 fibroblast growth factors (FGFs) (1), which have essential roles in metabolism and development. FGFs have been identified to be involved in processes of embryogenesis, including gastrulation, somitogenesis, body plan formation and organogenesis, as well as in skin wound healing (2-7). Initially, 1, a member of the FGF family, was reported to be preferentially expressed in the liver (8). However, recent studies have identified that FGF21 is inducible by starvation or certain drugs, and have reported on its diverse functions in glucose homeostasis as well as hepatoand cardioprotection (9-11). FGF19, FGF21 and FGF23 belong to the FGF19 sub-family. Among them, FGF21 primarily activates FGF receptor (FGFR)1c, for which co-receptor β-klotho is required (12,13).Previous efforts to produce recombinant human FGF21 (rhFGF21) using an Escherichia (E.) coli system resulted in low expression of soluble protein, indicating that the majority of recombinant protein localized in inclusion bodies. Although the small ubiquitin-like modifier (SUMO) fusion system has been shown to facilitate the soluble expression and enhance the production of bioactive rhFGF21 (14), additional steps are required for the removal of tags from the protein expressed in vitro. At present, Pichia (P.) pastoris (yeast) is among the most successful eukaryotic protein expression systems. Compared with mammalian cells, culture of P. pastoris requires simple and cheap growth media and conditions, and P. pastoris is known to...

TCF4/β‑catenin complex is directly upstream of FGF21 in mouse stomach cancer cells

“…FGF21 is sensitive to diverse physiological changes (e.g., fasting and injury) and serves an important role in development and metabolism, including glucose metabolism (11)(12)(13)(14)(15). Transcription of FGF21 was previously identified under the control of PPARα (17), RORα (18), and RARβ in hepatocytes (19).…”

“…In order to examine whether TCF4 directly binds to the putative motifs, ChIP assays were performed using the TCF4 antibody and the precipitation without antibody as the control. FGF21 exhibited a higher expression level in the MFC cells (data not shown); therefore, the MFC cells were used in the experiments (14,15). The IP DNA fragments were amplified by two primer pairs, which spanned the F1 and F2 regions in the FGF21 promoter (Fig.…”

“…FGF21 has been reported to preferentially express in the liver (10). However, previous studies have identified that FGF21 is also highly expressed in mouse skin tissue as well as an inducible feature of FGF21 by starvation, drug administration and wounding, and its diverse function in glucose homeostasis, liver and heart protection from injury or skin wound healing (11)(12)(13)(14)(15). Recently, FGF21 has been shown to improve hepatic insulin sensitivity by inhibiting the mammalian target of the rapamycin complex 1 (16).…”

Prokaryotic soluble expression and purification of bioactive human fibroblast growth factor 21 using maltose-binding protein

“…Given the diverse roles of FGF21, it is a promising therapeutic agent for the treatment of metabolic syndrome 5 , 7 , 8 . Previous attempts to produce human FGF21 (hFGF21) have mandated high cost approaches that nevertheless result in suboptimal recovery of bioactive protein 3 , 9 , 10 .…”

“…In another study, hFGF21 was expressed in soluble form under optimised fermentation conditions; however, the protein was purified without removal of its hexahistidine tag 15 . Eukaryotic production platforms allow for soluble expression of hFGF21 9 , 16 , however, the downside is poor productivity, protein hyper-glycosylation, and proteolysis at the N terminus of the peptide 16 . For optimizing the efficient production of the native hFGF21 protein, here we studied the ability of tag sequences to enhance the expression and solubility of FGF21 in E. coli .…”