Tissue transglutaminase has been identified as a contributor to a wide variety of diseases, including cataract formation and Celiac disease. Guinea pig tissue transglutaminase has a very broad substrate specificity and therefore is useful for kinetic studies using substrate analogues. Here, we report the expression in Escherichia coli of a hexahistidine-tagged guinea pig liver tissue transglutaminase (His(6)-tTGase) allowing rapid purification by immobilized-metal affinity chromatography. Using this procedure we have obtained the highest reported specific activity (17 U/mg) combined with a high yield (22 mg/L of culture) for recombinant TGase using a single-step purification protocol. Using two independent spectrophotometric assays, we determined that the Km value of the recombinant enzyme with the substrate Cbz-Gln-Gly is in the same range as values reported in the literature for the native enzyme. We have thus developed a rapid and reproducible protocol for the preparation of high quality tissue TGase. (C) 2003 Elsevier Inc. All rights reserved.