ABSTRACT
Fibroblast growth factor 17 (FGF17) is a novel member of the FGFs family, which is essential for cell development, tissue repair, tumor growth and invasion. The aim of the current study was to obtain a high expression level of recombinant human FGF17 (rhFGF17), including soluble proteins and inclusion bodies. An optimized rhFGF17 cDNA sequence was cloned into a pET3a vector, then the pET3ahFGF17 vector was transformed into BL21(DE3)pLysS Escherichia coli cells. Expression was induced by optimizing the conditions using isopropyl ßD1thiogalactopyranoside (IPTG) and it was confirmed that a 24h exposure to 0.8 mM IPTG at 16ËC provided the optimal condition for soluble hFGF17. Furthermore, for the inclusion bodies, the optimal condition was a 4h exposure to 0.4 mM IPTG at 37ËC. Two forms of rhFGF17 protein were purified by heparin affinity and SP Sepharose Fast Flow chromatography. MTT assays demonstrated that the purified rhFGF17 exerted an important effect on the proliferative activity of NIH3T3 cells, although there was no significant difference when compared with standard rhFGF17. Thus, an optimal and economic expression system was created in the present study for rhFGF17 in E. coli. This expression strategy enables the preparation of sufficient and highly bioactive rhFGF17 for further investigation of underlying mechanisms.
Subject(s)
Escherichia coli/genetics , Escherichia coli/metabolism , Fibroblast Growth Factors/biosynthesis , Fibroblast Growth Factors/genetics , Recombinant Proteins , Animals , Cell Proliferation/drug effects , Fibroblast Growth Factors/isolation & purification , Fibroblast Growth Factors/pharmacology , Gene Expression , Gene Order , Genetic Vectors/genetics , Humans , Mice , NIH 3T3 CellsABSTRACT
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 Aß25-35-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.
Subject(s)
Apoptosis Regulatory Proteins/genetics , Endoplasmic Reticulum Stress/drug effects , Fibroblast Growth Factors/biosynthesis , Neuroprotective Agents/metabolism , Recombinant Fusion Proteins/biosynthesis , Amyloid beta-Peptides , Animals , Apoptosis/drug effects , Apoptosis Regulatory Proteins/metabolism , Chemical Precipitation , Cloning, Molecular , Escherichia coli/genetics , Escherichia coli/metabolism , Fibroblast Growth Factors/genetics , Fibroblast Growth Factors/pharmacology , Gene Expression Regulation , Humans , Inclusion Bodies/chemistry , Mice , NIH 3T3 Cells , Neuroprotective Agents/chemistry , Neuroprotective Agents/pharmacology , PC12 Cells , Peptide Fragments , Plasmids/chemistry , Plasmids/metabolism , Protein Refolding , Rats , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/pharmacology , SolubilityABSTRACT
Human fibroblast growth factor 8b (FGF8b) was expressed based on a baculovirus expression vector system (BEVS) and identified as having a protective effect on Parkinson's disease. Immunoblotting demonstrated that rhFGF8b proteins were recognized by a human anti-FGF8b antibody. The multiplicity of infection and timing of harvest had a significant effect on protein yield and protein quality. Our results indicated that the rhFGF8b was first detectable at 36 h postinfection and reached a maximum at 60 h. A multiplicity of infection (MOI) of 8 pfu/mL was suitable for harvest. The target protein was purified by heparin-affinity chromatography. In vitro methylthiazol tetrazolium (MTT) assays demonstrated that the purified rhFGF8b could significantly stimulate proliferation of NIH3T3 cells. Furthermore, to elucidate the effect of rhFGF8b on Parkinson's disease, we used FGF8b pretreatment on a cell model of Parkinson's disease. The results indicated that rhFGF8b prevented necrosis and apoptosis of 1-METHYL-4-phenyl pyridine (MPP(+)) treated PC12 cells. Moreover, the effect of FGF8b on messenger RNA (mRNA) levels of apoptosis and ERS genes was investigated to clarify the molecular mechanisms of FGF8b. The results suggest that FGF8b exerts neuroprotective effects by alleviating endoplasmic reticulum (ER) stress during PD. These results suggest that FGF8b may be a promising candidate therapeutic drug for neurodegenerative diseases related to ER stress.
