Construction, expression, and characterization of recombinant hirudin in Escherichia coli

The mutant gene of HV2-K47 was obtained by polymerase chain reaction-directed mutagenesis and expressed in Escherichia coli. Many elements that could affect its expression level were compared. The product was purified to homogeneity via three chromatographic steps—ion exchange, gel filtration, and reverse phase chromatography—on the AKTA Explorer System. The antithrombin activity of HV2-K47 is much higher than that of recombinant HV2. Some properties and expression conditions were investigated systematically, which would be useful for further studies of hirudin and other small proteins.