Food Biotechnology, 26:143–153, 2012 Copyright © Taylor & Francis Group, LLC ISSN: 0890-5436 print / 1532-4249 online DOI: 10.1080/08905436.2012.670829 Cloning, Expression, Purification and Refolding of Caprine Prochymosin Mohammad Hadi Eskandari 1 , Arsalan Hosseini 2 , Sepideh Alasvand Zarasvand 1 , and Mahmoud Aminlari 3 1 Department of Food Science and Technology, College of Agriculture, Shiraz University, Shiraz, Iran 2 Department of Pathobiology, School of Veterinary Medicine, Shiraz University, Shiraz, Iran 3 Department of Biochemistry, School of Veterinary Medicine, Shiraz University, Shiraz, Iran Chymosin is an aspartic proteinase found in the stomach of neonatal mammals and is used as milk coagulant in the cheese industry. In this study, preprochymosin cDNA from the abomasums of Iranian natives’ kid goats was cloned and characterized. This cDNA has an open reading frame of 1143 bp and is predicted to code for a preproenzyme of 381 amino acids with an N-terminal 16 amino acid signal peptide that is followed by a 42 amino acid proenzyme. The deduced amino acid sequence revealed 98.7% and 94% identity with corresponding lamb and cattle sequences, respectively. The cDNA encod- ing for prochymosin was then subcloned into pET-28a and expressed with and without N-terminal His-tag in E. coli BL 21 (DE 3 ). Inclusion bodies were isolated and solubilized in 8 M urea at pH 10.7. Solubilized prochymosion molecules were successfully refolded and active recombinant goat chymosin was recovered after subsequent activation. Milk clotting activity (416 U/mg and 192 U/mg) was observed in (His + )-prochymosin and (His - )-prochymosin enzymes, respectively, after activation. Key Word: caprine; recombinant prochymosin; His-tag; activity INTRODUCTION Chymosin (rennin; E.C. 3.4.23.4), an aspartic proteinase normally present in the stomach of all nursing mammals and is used as the major milk clotting enzyme in the cheese industry (Chitpinityol and Crabbe, 1998). Chymosin is secreted as a zymogen called prochymosin, which consists of a single Address correspondence to Mohammad Hadi Eskandari, Department of Food Science and Technology, College of Agriculture, Shiraz University, Shiraz, Iran; E-mail: eskandar@shirazu.ac.ir