Fibroblast growth factor -21 (FGF-21) is a recently discovered metabolic regulation factor, regulating glucose and lipid metabolism and increasing insulin sensitivity. FGF-21 is expected to be a potential anti-diabetic drug. Expression of FGF-21 as inclusion bodies has advantages for high yield and purity, but the bioactivity of the protein is almost totally lost after denature and renature. That is why FGF-21 is currently expressed in soluble form. As a result, the yield is considerably low. In this study, we used SUMO vector to express SUMO-human FGF-21 (SUMO-hFGF-21) in form of inclusion body. We optimized the culture conditions to increase the yield of the bioactive human fibroblast growth factor-21. We applied the hollow fiber membrane filtration column to enrich the bacteria, wash, denature and renature inclusion bodies. After affinity and gel filtration chromatography, we examined the hypoglycemic activity of FGF-21 by the glucose uptake assay in HepG2 cells. We also detected the blood glucose concentration of type 2 diabetic db/db model mice after short or long-term treatment. The results show that the yield of ihFGF-21 was 4 times higher than that of shFGF-21. The yield was 20 mg/L for ihFGF-21 vs. 6 mg/L for shFGF-21. The purity of ihFGF-21 was above 95%, while shFGF-21 was 90%. Compared with the traditional method of extracting inclusion bodies, the production cycle was about three times shortened by application of hollow fiber membrane filtration column technology, but the bioactivity did not significantly differ. This method provides an efficient and cost-effective strategy to the pilot and industrial production of hFGF-21.
Animals ; Bacteria ; metabolism ; Diabetes Mellitus, Experimental ; drug therapy ; Disease Models, Animal ; Fibroblast Growth Factors ; biosynthesis ; Genetic Vectors ; Glucose ; metabolism ; Hep G2 Cells ; Humans ; Hypoglycemic Agents ; isolation & purification ; Inclusion Bodies ; metabolism ; Mice ; Recombinant Fusion Proteins ; biosynthesis ; Small Ubiquitin-Related Modifier Proteins ; biosynthesis

Objective To construct bispecific antibody BsAb1/17 against both IL-1β and IL-17A,express and purify the biologically active BsAbl/17 protein in prokaryotic system for further studies and applications. Methods VH1VL17-CL and VL1VH17-CH1 gene segments were constructed by overlap-PCR.Restriction enzyme sites Nco Ⅰ and BamH Ⅰ were designed at the 5'and 3' end primers respectively. The products of overlap-PCR were ligated to the Nco Ⅰ/BamH Ⅰ -prepared pET-27b vector. The recombinant plasmids pET-27b-VH1 VL17-CL(petA) and pET-27b-VL1 VH17-CH1 ( petB ) were transformed into E. coliRosetta separately. The expressing products were analyzed by SDS-PAGE and Western blot. Neutralization activity of the bispecific antibody for blocking the induction of IL-18 expression by IL-1β in human T cells was determined by real-time PCR. Neutralization activity of the bispecific antibody for blocking the induction of IL-6 expression by IL-17A in HeLa cells was determined by ELISA assay. Results The structure of the plasmids pET-27b-VH1 VL17-CL(petA) and pET-27b-VL1 VH17-CH1 (petB)was confirmed by DNA sequencing. After induction, the fusion proteins were expressed mainly as inclusion bodies. The purity of the both proteins exceeded 90%. SDS-PAGE analysis suggests the relative molecular mass of both products expressed by petA and petB were approximately 38× 103, which is in accordance with the theoretical value. The results of Western blot and ELISA test demonstrated that BsAb1/17 molecule had binding ability to both IL-1β and IL-17A. The BsAb1/17 could block IL-1β to stimulate human T cell to express IL-18 and block IL-17A to stimulate HeLa cell to express IL-6. Conclusion We successfully constructed a novel bispecific antibody BsAb1/17 against both IL-1 β and IL-17A, and expressed biologically active BsAb1/17 protein in prokaryotic system.

Objective To express the anti-IL-1βscfv and soluble TNF receptor 1 (sTNFR1),and analyze their bio-activities.Methods sTNFR1 was obtained by RT-PCR from the total RNA of HeLa cells,and fused with IL-1βscfv by the hinge fragment of IgG molecule.The fusion gene IL-1scfv:TNFR1 was cloned into the expression vector pET27b(+).The fusion protein was expressed and purified from inclusion bodies.Results The ELISA analysis showed that the fusion protein could bind hIL-1β and hTNF-α respectively in a dose-dependent manner,indicating that scfv and sTNFR in the fusion protein can form the correct spatial configuration.The dolt-blot analysis showed that the fusion protein could concurrently bind with hIL-1β and hTNF-α,indicating that the combination of the two parts of the fusion protein does not influence each other for binding to their target molecules.The bioactivity assay showed that the fusion protein could inhibit both the cytotoxicity of hTNF-α on L929 cells and hIL-1β-induced proliferation of L929 cells,indicating that the fusion protein has the ability to neutralize hTNF-α and hIL-1β.Conclusion A bispecific fusion protein IL-1scfv:TNFR1 was successfully constructed.The fusion protein has the ability to inhibit the biological activity of hTNF-α and hIL-1β,and provides a drug candidate for the treatment of rheumatoid arthritis.

The aim of this project is to establish a fibroblast growth factor-21 (FGF-21) signaling pathway targeted cell model, for screening a class of FGF-21 receptor agonists as anti-diabetic candidates. FGF-21 requires beta klotho transmembrane proteins as co-receptor for the activation of tyrosine kinase FGF receptor (FGFR) signaling, thereby activating a series of intracellular signaling pathways and regulating gene transcription for glucose metabolism. Firstly a recombinant plasmid expressing co-receptor beta klotho and EGFP reporter genes was constructed. After introducing the recombinant plasmid into package cells, the cell culture supernatant was used to infect 3T3-L1 cells, which were then screened for stably expressing beta klotho gene. Administration of FGF-21 increased the expression of GLUT1 and stimulated GLUT1-mediated glucose uptake. This novel cell model can be conveniently used in high-throughput drug screening of FGF-21 or FGF-21 analogues.

FGF21 (fibroblast growth factor 21) is a recently described member of the FGF family. It has been previously demonstrated that FGF21 is a potent regulator of glucose homeostasis. To improve stability of FGF21 for better efficacy, a new form of recombinant FGF21 was generated by fusion of a full length FGF21 gene and the Fc fragment of human IgG4 with flexible linker sequence. To examine the glucose regulation activity of FGF21-L-Fc, 3T3-L1 pre-adipocytes were differentiated into adipocytes, and glucose uptake activity of FGF21-L-Fc was examined by glucose oxidase and peroxidase (GOD-POD) assay. The results showed that in comparison with wild type FGF21, FGF21-L-Fc was more potent in stimulation of glucose uptake by 3T3-L1. In vivo studies on the modified protein demonstrated that FGF-L-Fc had a better efficacy in lowering blood glucose of the STZ-induced diabetic animals and controlled glucose level for a longer time. The results provided a sound basis for further studies.

Insulin resistance in insulin sensitive organ results in metabolic disorder such as hyperglycemia, hyperinsulinemia and hyper triglyceridemia which are common features of type 2 diabetes.Insulin resistance in liver cells mainly causes impaired glycogen synthesis, failed to suppress glucose production which is the major contribution to hyperglycemia.FGF-21 as a new metabolic regulator can control fasting blood glucose.The mechanism of FGF-21 effects on regulating plasma glucose has little to known.In order to establish an in vitro insulin resistant model of liver cells and evaluate the effects and mechanism of FGF-21 on glucose metabolism in the cell model, HepG2 cells were incubated with 10-7 mol/L insulin for 24 h to build insulin-resistant cell model.To evaluate the cells for insulin resistance, the cells were stimulated with fresh insulin for 24 h and the glucose uptake by these cells was carried out.The insulin-resistant cells were treated with different concentrations of FGF-21 for 24 h and insulin-treated cells were used as a control.The glucose uptake by the cells was detected by the method of glucose oxidizes/peroxides(GOD-POD);the synergy between insulin and FGF-21 was evaluated.The mRNA expression of GLUT1 in the insulin-resistant cells was detected by the real-time PCR.Glycogen synthesis of the cells was examined by the anthrone method.The results showed that HepG2 cells treated with 10-7 mol/L insulin for 24 h became resistant to insulin and the insulin resistance status was maintained for 48 h without change of cell morphology.FGF-21 could stimulate glucose consumption of the insulin-resistant model in a dose-dependent manner.The glucose consumption and glycogen synthesis of the insulin-resistant model were significantly improved by FGF-21 treatment.FGF-21 showed strong synergy with insulin in glucose uptake and glycogen synthesis of the model cells.While the cells became resistant to insulin, FGF-21 could increase the mRNA expression of GLUT1.Thus, It is concluded that FGF-21 stimulates glucose uptake in insulin resistant HepG2 cells through GLUT1 expression, stimulates glycogen synthesis and improves the glucose metabolism in the insulin resistant liver cell model.

This study is to evaluate the therapeutic effect of fibroblast growth factor 21 (FGF21) on type 2 diabetic mice model and to provide mechanistic insights into its therapeutic effect. Type 2 diabetic animal model was established with high calorie fat diet and low dose streptozotocin (STZ) injection. Mice were then randomized into 5 groups: model control, FGF21 0.25 and 0.05 μmol x kg(-1) x d(-1) groups, insulin treatment group. Ten age-matched normal KM mouse administered with saline were used as normal controls. Serum glucose, insulin, lipid products and the change of serum and liver tissue inflammation factor levels between five groups of mouse were determined. The results showed that blood glucose, insulin, free fatty acids (FFAs), triglycerides, and inflammatory factor average FGF-21 of type 2 diabetes model group and normal control group were significantly higher (P < 0.01), while compared with insulin group, no difference was significant. Average blood glucose, insulin, blood lipid and inflammatory factor of FGF-21 treatment group compared with type 2 diabetes group was significantly lower (P < 0.01) and insulin group has no difference with the model control group. The results of OGTT and HOMA-IR showed that insulin resistance state was significantly relieved in a dose-dependent manner. Thus, this study demonstrates that FGF-21 significantly remits type 2 diabetic mice model's insulin resistance state and participates in the regulation of inflammatory factor levels and type 2 diabetes metabolic disorders.

Fibroblast growth factor-21 (FGF-21) is an important metabolism regulator, however, whether FGF-21 has effects on cardiovascular remains unclear. In this study, H2O2-induced injury in H9c2 cells was used as a cell model, the anti-apoptosis potential and mechanism of FGF-21 against oxidative injury were evaluated by MTT assay, flow cytometry assay and real-time PCR. The results showed that FGF-21 could increase the cell survival of H2O2-induced injury in H9c2 cells and prevent H9c2 cells from oxidative stress-induced apoptosis. Furthermore, FGF-21 can elevate SOD activity and regulate Bcl-2/Bax expression in H9c2 cells. The results suggest that FGF-21 have protective effect against the H2O2-induced apoptosis in H9c2 cells.

The aim of the study is to establish a platform to deliver therapeutic proteins into target cells through a polyarginine-based cell penetrating peptide. To facilitate the expression of therapeutic proteins, a pSUMO (Small Ubiquitin-like Modifier)-R9-EGFP (enhanced green fluorescence protein) prokaryotic expression vector was constructed. After induction, the fusion protein SUMO-R9-EGFP was efficiently expressed. To validate the cell penetrating ability of the fusion protein, HepG2 cells were incubated with the purified R9-EGFP or EGFP protein as control, internalization of the fluorescent proteins was examined by either flow cytometry or confocal microscopy. The result obtained by flow cytometry showed that the R9-EGFP fusion protein could efficiently penetrate into the HepG2 cells in a dose and time-dependent manner. In contrast, the fluorescence was barely detected in the HepG2 cells incubated with EGFP control. The fluorescence intensity of the R9-EGFP treated cells reached plateau phase after 1.5 h. The result obtained by confocal microscopy shows that R9-EGFP efficiently entered into the HepG2 cells and was exclusively located in the cytoplasm, whereas, no fluorescence was detected in the cells incubated with the EGFP control. The heparin inhibition experiment showed that heparin could inhibit penetrating effect of the R9-EGFP protein by about 50%, suggesting that the penetrating ability of the fusion protein is heparin-dependent. In summary, the study has established a platform to deliver therapeutic proteins into target cells through a polyarginine-based penetrating peptide.
Cell-Penetrating Peptides ; biosynthesis ; genetics ; pharmacology ; Genetic Vectors ; genetics ; Green Fluorescent Proteins ; biosynthesis ; genetics ; Hep G2 Cells ; Humans ; Peptides ; genetics ; metabolism ; Protein Transport ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; pharmacology