BACKGROUND: Antithrombin III (AT III) is a serine protease inhibitor that inhibits thrombin and the activated forms of factors X, VII, IX, XI and XII. Transgenic expression of therapeutic proteins in animal systems has gradually matured from laboratory scale to industrial practice, demanding efficient and scalable purification processes. The purification and characterization of recombinant human antithrombin III (rhAT III) from transgenic goat milk are described here. RESULTS: The rhAT III was purified by isoelectric precipitation, heparin affinity chromatography, and size exclusion chromatography, resulting in a 90.6% yield and >99% purity. The goat beta-casein secretion peptide introduced to the rhAT III was cut off using enterokinase and removed by size exclusion chromatography using a Superdex 75 column. The primary structure, disulfide linkages, glycosylation sites, secondary structure and tertiary structure of the rhAT III were measured and found to be the same as those of the plasma-derived AT III (phAT III). CONCLUSION: A facile process is introduced for the purification of rhAT III from transgenic goat milk. The rhAT III with high purity was achieved after an initial isoelectric precipitation step in which most of the bulk protein impurities are removed, followed by affinity chromatography and size exclusion chromatography. The rhAT III was demonstrated to have the same structure as phAT III. (C) 2011 Society of Chemical Industry