The cystatin superfamily encompasses proteins that contain multiple cystatin-like sequences. Some of the members are active cysteine protease inhibitors, while others have lost or perhaps never acquired this inhibitory activity. There are three inhibitory families in the superfamily, including the type 1 cystatins (stefins), type 2 cystatins and the kininogens. The cystatin locus on chromosome 20 contains the majority of the type 2 cystatin genes and pseudogenes. CST3 is located in the cystatin locus and is the most abundant extracellular inhibitor of cysteine proteases, which is found in high concentrations in biological fluids and is expressed in virtually all organs of the body. Recombinant human CST3 protein, fused to His-tag at N-terminus, was expressed in E.coli and purified by using conventional chromatography techniques.