ABSTRACT
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.
Subject(s)
Oxyntomodulin/biosynthesis , Recombinant Proteins/biosynthesis , Amino Acid Sequence , Catalysis , DnaB Helicases/biosynthesis , DnaB Helicases/genetics , Escherichia coli/genetics , Inteins/genetics , Molecular Sequence Data , Mutation , Oxyntomodulin/genetics , Oxyntomodulin/isolation & purification , Plasmids/chemistry , Plasmids/genetics , Recombinant Fusion Proteins/biosynthesis , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/isolation & purification , Recombinant Proteins/genetics , Recombinant Proteins/isolation & purification , Synechocystis/enzymology , Synechocystis/geneticsABSTRACT
Human brain natriuretic peptide (hBNP) was used clinically for the treatment of acute decompensated congestive heart failure. In this paper, hBNP was expressed as a fusion protein with a histidine tag and Ssp dnaB mini-intein which was capable of self-cleavage. After affinity chromatography with Ni-Sepharose and renaturation, the fusion protein was enriched with CM-cellulose. Ssp dnaB mini-intein mediated peptide-bond hydrolysis was triggered by shifting the pH and temperature in the CM-cellulose column, which let to the release of hBNP from the fusion protein and the separation of hBNP from His-DnaB. The hBNP sample was further purified by C4 reverse phase HPLC, and 2.8mg of the peptide with homogeneity of 97% was obtained from one liter of culture medium. The biological activity was assayed in vitro, which indicated that hBNP had a potent vasodilatory effect on rabbit aortic strips with an EC50 of 1.94 x 10(-6) mg/mL.
