title: High production in E. coli of biologically active recombinant human fibroblast growth factor 20 and its neuroprotective effects.
abstract: Fibroblast growth factor 20 (FGF20) has a wide range of biological activities; its expression is most pronounced in neural tissues where it has functions in development and neuroprotection. Given these activities, interest in the clinical applications of FGF20 is rising, which will lead to increasing demand for active recombinant human FGF20 (rhFGF20). To improve the production of rhFGF20, an artificial gene encoding fgf20 was cloned into pET3a and expressed in E. coli BL21(DE3)pLysS. By optimizing induction conditions, we successfully induced large amounts of insoluble rhFGF20. Following solubilization and refolding of the rhFGF20 from inclusion bodies, it was purified by HiTrap heparin affinity chromatography to a purity of over 96% with a yield of 218 mg rhFGF20/100 g wet cells. The purified rhFGF20 could stimulate proliferation of both NIH 3T3 cells and PC-12 cells, measured by the MTT assay. In a model of Aβ25-35-induced apoptosis on PC-12 cells, rhFGF20 had a clear protective effect. RT-PCR and Western blot analysis of apoptosis-related genes and proteins revealed that the FGF20-derived protective mechanism was likely due to the relief of endoplasmic reticulum stress (ER stress). In conclusion, the approach described here may be a better means to produce active rhFGF20 in good quantity, thereby allowing for its future pharmacological and clinical use.
authors.affiliation: School of Pharmaceutical Science, Wenzhou Medical University, Chashan College Park, Wenzhou, 325035, China.
authors.forename: Haishan
authors.initials: H
authors.lastname: Tian
authors.affiliation: School of Pharmaceutical Science, Wenzhou Medical University, Chashan College Park, Wenzhou, 325035, China.
authors.forename: Yang
authors.initials: Y
authors.lastname: Zhao
authors.affiliation: School of Pharmaceutical Science, Wenzhou Medical University, Chashan College Park, Wenzhou, 325035, China.
authors.forename: Nazi
authors.initials: N
authors.lastname: Chen
authors.affiliation: School of Pharmaceutical Science, Wenzhou Medical University, Chashan College Park, Wenzhou, 325035, China.
authors.forename: Meiyu
authors.initials: M
authors.lastname: Wu
authors.affiliation: School of Pharmaceutical Science, Wenzhou Medical University, Chashan College Park, Wenzhou, 325035, China.
authors.forename: Weiyue
authors.initials: W
authors.lastname: Gong
authors.affiliation: School of Pharmaceutical Science, Wenzhou Medical University, Chashan College Park, Wenzhou, 325035, China.
authors.forename: Jie
authors.initials: J
authors.lastname: Zheng
authors.affiliation: School of Pharmaceutical Science, Wenzhou Medical University, Chashan College Park, Wenzhou, 325035, China.
authors.forename: David G
authors.initials: DG
authors.lastname: Fernig
authors.affiliation: School of Pharmaceutical Science, Wenzhou Medical University, Chashan College Park, Wenzhou, 325035, China.
authors.forename: Alois
authors.initials: A
authors.lastname: Jungbauer
authors.affiliation: School of Pharmaceutical Science, Wenzhou Medical University, Chashan College Park, Wenzhou, 325035, China.
authors.forename: Dezhong
authors.initials: D
authors.lastname: Wang
authors.affiliation: School of Pharmaceutical Science, Wenzhou Medical University, Chashan College Park, Wenzhou, 325035, China. proflxk@163.com.
authors.forename: Xiaokun
authors.initials: X
authors.lastname: Li
authors.affiliation: School of Pharmaceutical Science, Wenzhou Medical University, Chashan College Park, Wenzhou, 325035, China. chaojiang10@hotmail.com.
authors.forename: Chao
authors.initials: C
authors.lastname: Jiang
mesh_terms: D016229:Amyloid beta-Peptides; D000818:Animals; D017209:Apoptosis / Q000187:drug effects; D051017:Apoptosis Regulatory Proteins / Q000235:genetics* / Q000378:metabolism; D011232:Chemical Precipitation; D003001:Cloning, Molecular; D059865:Endoplasmic Reticulum Stress / Q000187:drug effects*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005346:Fibroblast Growth Factors / Q000096:biosynthesis* / Q000235:genetics / Q000494:pharmacology; D005786:Gene Expression Regulation; D006801:Humans; D002479:Inclusion Bodies / Q000737:chemistry; D051379:Mice; D041681:NIH 3T3 Cells; D018696:Neuroprotective Agents / Q000737:chemistry / Q000378:metabolism* / Q000494:pharmacology; D016716:PC12 Cells; D010446:Peptide Fragments; D010957:Plasmids / Q000737:chemistry / Q000378:metabolism; D058849:Protein Refolding; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000494:pharmacology; D012995:Solubility
chemical_list: D016229:Amyloid beta-Peptides; D051017:Apoptosis Regulatory Proteins; C416003:FGF20 protein, human; D018696:Neuroprotective Agents; D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; C074636:amyloid beta-protein (25-35); D005346:Fibroblast Growth Factors
keywords: Inclusion body protein; Mitogenic activity; PC-12 cells; Purification; rhFGF20