Recombinant oxyntomodulin

An artificial gene encoding oxyntomodulin was obtained using chemical and enzymatic methods and cloned into Escherichia coli. A recombinant plasmid was constructed containing a hybrid oxyntomodulin gene and Ssp dnaB intein from Synechocystis sp. The expression of the resulting hybrid gene in E. coli, its properties, and the conditions of its autocatalytic cleavage to oxyntomodulin were studied.     

Recombinant fragment 44–77 of the pigment epithelium-derived factor prevents the development of the pathological cornea neovascularization

The pigment epithelium-derived factor (PEDF), a secreted 50 kDa glycoprotein, is one of the most potent endogenous inhibitors of angiogenesis. The fragment 44–77 of PEDF possesses the antiangiogenic properties of the full-sized protein and is a potential drug candidate for the treatment of diseases of visual organs accompanied by pathological neovascularization. An effective biotechnological method for the large-scale production of the PEDF (44–77) fragment as part of the fusion protein with the SspDnaB intein has been developed. The hybrid protein was produced in Escherichia coli bacterial cells in the form of inclusion bodies, solubilized, and subjected to autocatalytic cleavage with the release of the PEDF (44–77) fragment (reaction yield 77%). The target peptide was separated from the intein by 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 l of culture. The antiangiogenic activity of the peptide was confirmed in vitro using mouse endothelial cells SVEC-4-10. It was found that the preparation at a concentration of 1 nM suppresses the proliferation of endothelial cells by 53% and inhibits the formation of tube-like structures by endothelial cells. The ability of the recombinant PEDF (44–77) to block the initial stages of angiogenesis was shown using an experimental model of rabbit corneal neovascularization.     

Recombinant oxyntomodulin

An artificial gene encoding oxyntomodulin was obtained using chemical and enzymatic methods and cloned into Escherichia coli. A recombinant plasmid was constructed containing a hybrid oxyntomodulin gene and Ssp dnaB intein from Synechocystis sp. The expression of the resulting hybrid gene in E. coli, its properties, and the conditions of its autocatalytic cleavage to oxyntomodulin were studied.     

Recombinant fragment of pigment epithelium-derived factor (44-77) prevents pathological corneal neovascularization

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.     

Production of recombinant oxytocin through sulfitolysis of inteincontaining fusion protein

An artificial gene consisting of seven copies of an oxytocinoyl-lysine encoding sequence arranged in a tandem was synthesized and inserted downstream of the SspDnaB intein gene in a pTWIN1 plasmid. The corresponding fusion protein Dnab-7oxy contained 16 cysteine residues and formed inclusion bodies when expressed in E. coli. The standard protocol involving solubilization of the fusion protein and its autocatalytic cleavage on a chitin resin was not effective because of a very low yield of the cleavage reaction. Attempts to perform a refolding of the intein part of the fusion protein in solution were also unsuccessful because of a high level of protein aggregation. Sulfitolysis of cysteine residues is known to increase a solubility of proteins and peptides. Therefore we suggested a one-step approach that combines solubilization of inclusion bodies and sulfitolysis of a hybrid protein. The fusion protein was completely reduced and solubilized in 8M urea at pH 9.0 in the presence of sodium sulfite and sodium tetrathionate. The sulfitized protein was loaded onto a chitin column, an efficient cleavage was induced by a pH shift from 9.0 to 6.5, and seven successively connected oxytocinoyl- lysine units were released. The heptamer was subjected to trypsinolysis yielding sulfitized monomers of oxytocinoyllysine. Oxytocinoyl-lysine was refolded as described previously and treated by carboxypeptidase B to form the oxytocinic acid. The target oxytocin amide was then synthesized via methyl ester intermediate. Using this approach 6 mg of recombinant oxytocin can be obtained from 1 g of biomass.     

Production and purification of recombinant human glucagon overexpressed as intein fusion protein in Escherichia coli

Chemico-enzymatic synthesis and cloning in Esherichia coli of an artificial gene coding human glucagon was performed. Recombinant plasmid containing hybrid glucagons gene and intein Ssp dnaB from Synechocestis sp. was designed. Expression of the obtained hybrid gene in E. coli, properties of the formed hybrid protein, and conditions of its autocatalytic cleavage leading to glucagon formation were studied.