ABSTRACT
Ribokinase (RK) was expressed in the Escherichia coli ER2566 cells harboring the constructed expression plasmid encompassing the rbsK gene, encoding ribokinase. The recombinant enzyme was purified from sonicated cells by double chromatography to afford a preparation that was ca. 90% pure and had specific activity of 75 micromol/min mg protein. Catalytic activity of RK: (i) is strongly dependent on the presence of monovalent cations (potassium>>>ammonium>cesium), and (ii) is cooperatively enhanced by divalent magnesium and manganese ions. Besides D-ribose and 2-deoxy-D-ribose, RK was found to catalyze the 5-O-phosphorylation of D-arabinose, D-xylose, and D-fructose in the presence of ATP, and potassium and magnesium ions; L-ribose and L-arabinose are not substrates for the recombinant enzyme. A new radiochemical method for monitoring the formation of D-pentofuranose-5-[32P]phosphates in the presence of [gamma-32P]ATP and RK is reported.
Subject(s)
Escherichia coli/enzymology , Phosphotransferases (Alcohol Group Acceptor) , Binding Sites , Catalysis , Enzyme Activation , Kinetics , Nucleosides/chemical synthesis , Nucleosides/chemistry , Phosphotransferases (Alcohol Group Acceptor)/biosynthesis , Phosphotransferases (Alcohol Group Acceptor)/chemistry , Phosphotransferases (Alcohol Group Acceptor)/isolation & purification , Structure-Activity Relationship , Substrate SpecificityABSTRACT
The plasmid DNA pERilox4 containing the gene of the recombinant protein, which included the leader sequence and the oxytocinoyl lysine tetramer, was constructed. The high level of gene expression in E. coli was achieved. The method for purification of the recombinant protein and its isolation in the soluble form was developed. The conditions for digestion of the hybrid protein by trypsin and carboxypeptidase B were matched. The effective method for transformation of oxytocinic acid to oxytocin was worked out. The scheme suggested allowed obtaining oxytocin in high yield.
Subject(s)
Escherichia coli/genetics , Oxytocin/biosynthesis , Recombinant Fusion Proteins/biosynthesis , Amino Acid Sequence , Base Sequence , Chromatography, High Pressure Liquid/methods , Cloning, Molecular , Electrophoresis, Polyacrylamide Gel/methods , Gene Expression Regulation, Bacterial/genetics , Humans , Interleukin-3/genetics , Molecular Sequence Data , Oxytocin/chemistry , Oxytocin/genetics , Plasmids/genetics , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/isolation & purification , Transformation, BacterialABSTRACT
The Escherichia coli genes encoding purine nucleoside phosphorylase, uridine phosphorylase, and thymidine phosphorylase were cloned into pET plasmids to generate highly effective E. coli BL21(DE3) strains producing each of these enzymes. Optimum conditions for biosynthesis of each enzyme as a soluble protein with intact biological activity were found. The crude preparations are approximately 80% pure and can be used immediately for enzymatic transglycosylation. The enzyme preparations were purified to homogeneity by two steps including fractional precipitation with ammonium sulfate and subsequent chromatography on Sephadex G-100 and DEAE-Sephacel.
