RESUMO
The therapeutic efficiency of recombinant thymosin ß4 (rTß4) synthesized by us was studied in vivo on spontaneous CBRB mouse model that is adequate to human chronic dermatitis. Three applications of the drug during a week significantly alleviated symptoms of the disease in female mice, and in complex with subsequent antibacterial and antifungal therapy led to a pronounced and lasting (2 months) therapeutic effect. The results attest to a possibility of using rTß4 in combination with the known treatment protocols for chronic inflammatory diseases of the skin.
Assuntos
Dermatite/tratamento farmacológico , Proteínas Recombinantes/uso terapêutico , Timosina/uso terapêutico , Animais , Doença Crônica/tratamento farmacológico , Modelos Animais de Doenças , Feminino , CamundongosRESUMO
Pigment epithelium-derived factor (PEDF), a 50 kDa secreted glycoprotein, is among the most potent endogenous inhibitors of angiogenesis. PEDF-derived fragment (44-77) possesses antiangiogenic properties of the full-sized protein and is a potential drug candidate for the treatment of ocular neovascular diseases. In this study we propose an efficient scalable biotechnological method for the production of PEDF (44-77) as part of a fusion protein with SspDnaB intein. The fusion protein was obtained in bacterial E. coli cells in the form of inclusion bodies, solubilized and subjected to autocatalytic cleavage with the release of PEDF (44-77) (yield, 77%). The target peptide was separated from the intein using tangential ultrafiltration. The final purification of PEDF (44-77) was performed by reversed-phase HPLC. The yield of the target peptide (purity, 99%) was 65 mg per 1 liter of culture. Antiangiogenic activity of the obtained peptide was studied in vitro using murine endothelial cells SVEC-4-10. PEDF (44-77) suppressed proliferation of endothelial cells by 53% and inhibited endothelial cell tube formation at the concentration of 1 nM. The ability of the recombinant PEDF (44-77) to block initial stages of angiogenesis was demonstrated using the model of rabbit corneal neovascularization.
Assuntos
Inibidores da Angiogênese/biossíntese , Inibidores da Angiogênese/farmacologia , Neovascularização da Córnea/prevenção & controle , Proteínas do Olho/biossíntese , Proteínas do Olho/farmacologia , Fatores de Crescimento Neural/biossíntese , Fatores de Crescimento Neural/farmacologia , Serpinas/biossíntese , Serpinas/farmacologia , Inibidores da Angiogênese/genética , Animais , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Neovascularização da Córnea/tratamento farmacológico , Neovascularização da Córnea/genética , Neovascularização da Córnea/metabolismo , Neovascularização da Córnea/patologia , Modelos Animais de Doenças , Células Endoteliais/metabolismo , Células Endoteliais/patologia , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas do Olho/genética , Humanos , Camundongos , Fatores de Crescimento Neural/genética , Coelhos , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética , Proteínas Recombinantes/farmacologia , Serpinas/genéticaRESUMO
Thymosin beta4 (43 aa) is a highly conserved acidic peptide which regulates actin polymerization in mammalian cells by sequestering globular actin. Thymosin beta4 is undergoing clinical trials as a drug for the treatment of venous stasis ulcers, corneal wounds and injuries, as well as acute myocardial infarction. Currently, thymosin beta4 is produced with solid-phase chemical synthesis. Biotechnological synthesis of this peptide presents difficulties because N-terminal amino acid residue of thymosin beta4 is acetylated. In this study we propose a method for producing the recombinant precursor of thymosin beta4 and its subsequent targeted chemical acetylation. Desacetylthymosin beta4 was synthesized as a part of a hybrid protein with thioredoxin and a specific TEV (tobacco etch virus) protease cleavage site. The following scheme was developed for the purification of desacetylthymosin beta4: (i) the biosynthesis of a soluble hybrid protein (HP) in Escherichia coli; (ii) isolation of the HP by ion exchange chromatography; (iii) cleavage of the HP with TEVprotease; (iv) purification of desacetylthymosin beta4 by ultra-filtration. N-terminal acetylation of desacetylthymosin beta4 was performed with acetic anhydride under acidic conditions (pH 3). The reaction yield was 55%. Thymosin beta4 was then purified by reverse-phase high performance liquid chromatography. The proposed synthetic approach to recombinant thymosin beta4 is suitable for scale-up and can provide for the medical use of highly purified preparation with a yield of 20 mg from 1 L of culture.
