Haishan Tian

High-Efficiency Expression of TAT-bFGF Fusion Protein in Escherichia coli and the Effect on Hypertrophic Scar Tissue

Background Basic fibroblast growth factor (bFGF) is a member of the fibroblast growth factor family that has effects on wounding healing and neuro-protection. However, it is difficult to use bFGF to treat diseases that are separated by physiological barriers, such as the dermal barrier and blood brain barrier. Methodology/Principal Findings To improve bFGF’s penetration ability, we fused the recombinant human fibroblast growth factor (rhbFGF) gene with TAT. We constructed a pET3c vector that contained the recombinant bFGF gene and successfully expressed this gene in the E. coli strain BL21 (DE3) pLsS. The fusion protein was purified using CM Sepharose FF and heparin affinity chromatography. The purity of the TAT-rhbFGF was greater than 95%, as detected by SDS-PAGE. An in vitro MTT trial revealed that the modified bFGF significantly promoted the proliferation of NIH3T3 cells. The cell penetration trial and the mouse skin penetration trial demonstrated that the fusion protein had certain penetration abilities. The animal experiments confirmed that TAT-rhbFGF was effective in the treatment of the hypertrophic scars. Conclusions/Significance We have successfully expressed and purified a TAT-rhbFGF fusion protein in this study. Our results have shown that the fusion protein had a greater ability to penetrate the dermal skin layer. TAT-rhbFGF improved the physical appearance of hypertrophic scars. TAT-rhbFGF may be a potential fusion protein in the treatment of dermal disorders, including hypertrophic scar.

Expression of bioactive recombinant human fibroblast growth factor 10 in Carthamus tinctorius L. seeds.

Fibroblast growth factor 10 (FGF10) is a member of the FGF superfamily. It exhibits diverse biological functions, and is extensively used for fundamental research and clinical applications involving hair growth, tissue repair, and burn wounds. Oil bodies, obtained from oil seeds, have been exploited for a variety of biotechnology applications. The use of oil bodies reduces purification steps and costs associated with the production of heterogonous proteins. Here, recombinant human FGF10 (rhFGF10) was expressed in safflower (Carthamus tinctorius L.) seeds using oilbody-oleosin technology. A plant expression vector, pOTBar-oleosin-rhFGF10, was constructed and introduced into safflower using Agrobacterium tumefaciens transformation, and mature safflower plants were obtained by grafting. Oleosin-rhFGF10 was successfully transformed and expressed in safflower seeds and inherited to the T3 generation. Moreover, MTT assays demonstrated that oil bodies expressed oleosin-FGF10 had a dose-dependent effect on cellular proliferation. In conclusion, this may provide a method of producing oleosin-rhFGF10, and help us meet the increasing pharmacological demands for the protein.

FGF21 Prevents Angiotensin II-Induced Hypertension and Vascular Dysfunction by Activation of ACE2/Angiotensin-(1–7) Axis in Mice

Fibroblast growth factor 21 (FGF21) is a metabolic hormone with pleiotropic effects on glucose and lipid metabolism and insulin sensitivity. However, the role of FGF21 in hypertension remains elusive. Here we show that FGF21 deficiency significantly exacerbates angiotensin II-induced hypertension and vascular dysfunction, whereas such negative effects are reversed by replenishment of FGF21. Mechanistically, FGF21 acts on adipocytes and renal cells to promote induction of angiotensin-converting enzyme 2 (ACE2), which in turn converts angiotensin II to angiotensin-(1-7), then inhibits hypertension and reverses vascular damage. In addition, ACE2 deficiency strikingly abrogates these beneficial effects of FGF21 in mice, including alleviation of angiotensin II-associated hypertension and vascular damage. Otherwise, pharmaceutical inhibition of angiotensin-(1-7) attenuates the protective effect of FGF21 on angiotensin II-induced vascular dysfunction, but not on hypertension. Thus, FGF21 protects against angiotensin II-induced hypertension and vascular impairment by activation of the ACE2/angiotensin-(1-7) axis via fine-tuning the multi-organ crosstalk between liver, adipose tissue, kidney, and blood vessels.

Fibroblast growth factor 9 subfamily and the heart

The fibroblast growth factor (FGF) 9 subfamily is a member of the FGF family, including FGF9, 16, and 20, potentially sharing similar biochemical functions due to their high degree of sequence homology. Unlike other secreted proteins which have a cleavable N-terminal secreted signal peptide, FGF9/16/20 have non-cleaved N-terminal signal peptides. As an intercellular signaling molecule, they are involved in a variety of complex responses in animal development. Cardiogenesis is controlled by many members of the transcription factor family. Evidence suggests that FGF signaling, including the FGF9 subfamily, has a pretty close association with these cardiac-specific genes. In addition, recent studies have shown that the FGF9 subfamily maintains functional adaptation and survival after myocardial infarction in adult myocardium. Since FGF9/16/20 are secreted proteins, their function characterization in cardiac regeneration can promote their potential to be developed for the treatment of cardioprotection and revascularization. Here, we conclude that the FGF9 subfamily roles in cardiac development and maintenance of postnatal cardiac homeostasis, especially cardiac function maturation and functional maintenance of the heart after injury.

Highly efficient production of functional recombinant human fibroblast growth factor 22 in E. coli and its protective effects on H2O2-lesioned L02 cells

In the 22 member mammalian FGF family, FGF22 belongs to FGF7 subfamily, and its effects are largely confined to the brain and skin. To explore the functions of FGF22 on other tissues and develop a large-scale production of recombinant human FGF22 (rhFGF22) without a fusion tag, a plasmid encoding human FGF22 (pET3a-rhFGF22) was used to express rhFGF22 in E. coli BL21 (DE3) pLysS. A large amount of rhFGF22 inclusion body protein was obtained. A two-step denaturing method successfully solubilized rhFGF22, and it was refolded and then purified in one step via heparin affinity chromatography. A yield of 105 mg rhFGF22 with a purity of up to 95% was obtained from 100 g wet bacteria. It was found that the rhFGF22 had biological activity, since it effectively attenuated H2O2-induced human hepatic L02 cell death. Analysis by qRT-PCR and Western blot demonstrated that rhFGF22 protects L02 cells from H2O2-induced oxidative damage via suppression of mitochondrial apoptosis pathways. In conclusion, the strategy described in this paper may provide a novel means to solve the production of insoluble rhFGF22 and shine new light on its translational potential.

Expression of Halo-hFGF18 and study of its effect on differentiation of ATDC5 cells

Fibroblast growth factor 18 (FGF18) is a member of the fibroblast growth factor family and important in cartilage growth and development. However, the mechanism by which FGF18 mediates its biological functions is still unclear. In our study, we expressed the rhFGF18 protein fused to a HaloTag, (Halo-rhFGF18). MTT assay results indicated that both rhFGF18 and Halo-rhFGF18 have similar biological activities in NIH3T3 cells. However, basic FGF and acidic FGF were more potent than both rhFGF18 and Halo-rhFGF18. Confocal imaging data indicated that the red fluorescence labeled Halo-rhFGF18 strongly bound to ATDC5 cells and stimulated their proliferation and differentiation, which suggests that glycosaminoglycans may be involved in mediating the biological effects of rhFGF18 in ATDC5 cells. Moreover, western blot results demonstrated that, in ATDC5 cells, ERK1/2 signaling is activated upon stimulation with rhFGF18. Our results may open doors for the use of rhFGF18 as a drug to promote cartilage growth.

Effective Protection on Acute Liver Injury by Halo Tag-Flanked Recombinant Fibroblast Growth Factor 7

The drug development of FGF7 has been restricted by its toxicity to the host, low expression, poor stability, and easy degradation. Recent studies have shown that Halo-tag-flanked recombinant human FGF7 can solve the problem of toxicity; however, its biological activity is unknown. This study aimed to explore the activity of Halo-rhFGF7 and rhFGF7 on acute liver injury in vitro and in vivo. The rhFGF7 is expressed with a N-terminal Halo-tag, followed by a tobacco etch virus (TEV) protease cleavage site, in Escherichia coli BL21 (DE3) pLysS in this study. The products could stimulate the proliferation of carbon tetrachloride-damaged L-O2 cells (normal human liver cells); they also inhibited cell apoptosis. Due to the use of the Halo, the protein could be tracked using fluorescence localization. Recombinant protein exerted a protective effect on the acute liver injury model in vitro and in vivo. The MTT assay and Western blot analysis showed that this protective effect is realized through various paths, including promoting proliferation, inhibiting cell apoptosis and anti-inflammatory. In conclusion, Halo-rhFGF7 and rhFGF7 displayed an excellent protective effect on acute liver injury. The present study provided an experimental basis and data support for further research on rhFGF7.

High-yield of biologically active recombinant human fibroblast growth factor-16 in E. coli and its mechanism of proliferation in NCL-H460 cells

ABSTRACT Fibroblast growth factor-16 (FGF16) is a member of FGF9 subfamily, which plays key role in promoting mitosis and cell survival, and also involved in embryonic development, cell growth, tissue repair, morphogenesis, tumor growth, and invasion. However, the successful high-yield purification of recombinant human fibroblast growth factor-16 (rhFGF16) protein has not been reported. In addition, lung cancer is a major cause of cancer-related deaths, which threats people’s lives and its incidence has continued to rise. Learning pathways or proteins, which involved in lung tumor progression will contribute to the development of early diagnosis and targeted therapy. FGF16 promoted proliferation and invasion behavior of SKOV-3 ovarian cancer cells, whose function may be similar in lung cancer. The hFGF16 was cloned into pET-3d and expressed in Escherichia coli BL21 (DE3) pLysS. Finally, obtained two forms of FGF16 that exhibited remarkable biological activity and the purity is over 95%, meanwhile, the yield of soluble 130 mg/100 g and insoluble 240 mg/100 g. Experiments demonstrated FGF16 could promote proliferation of NCL-H460 cells by activating Akt, Erk1/2, and p38 MAPK signaling, whereas JNK had no significant effect. In total, this optimized expression strategy enables significant quantity and activity of rhFGF16, thereby meeting its further pharmacological and clinical usages.