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.