tailieunhanh - Cloning, expression, and activity analysis of human cathepsin C in the yeast Pichia pastoris

The yeast Pichia pastoris expression system was investigated for the production of human cathepsin C (CatC) recombinant protein. The full-length CatC cDNA, corresponding to amino acids 12–475, was synthesized from interleukin-2 (IL-2) stimulated human peripheral blood mononuclear cells and subcloned in the pGEM-T cloning vector. | Turkish Journal of Biology Research Article Turk J Biol (2017) 41: 746-753 © TÜBİTAK doi: Cloning, expression, and activity analysis of human cathepsin C in the yeast Pichia pastoris Cenk DAĞLIOĞLU* Department of Molecular Biology and Genetics, Faculty of Science, İzmir Institute of Technology, Urla, İzmir, Turkey Received: Accepted/Published Online: Final Version: Abstract: The yeast Pichia pastoris expression system was investigated for the production of human cathepsin C (CatC) recombinant protein. The full-length CatC cDNA, corresponding to amino acids 12–475, was synthesized from interleukin-2 (IL-2) stimulated human peripheral blood mononuclear cells and subcloned in the pGEM-T cloning vector. After confirming the DNA sequence of the insert, the gene was cloned into the pPICZαA expression vector under the control of the methanol-inducible alcohol oxidase (AOX1) promoter and transformed to P. pastoris X-33 cells. The expressed protein was secreted into the culture medium through the α-factor mating signal sequence of the expression vector. Analysis of the culture supernatant revealed that the recombinant human CatC was secreted as a 58-kDa molecule, indicating that human CatC was accumulated in the culture supernatant as proform composed of the residual propart, the activation peptide, and the heavy and light chains. Extracellular recombinant proCatC was further activated by cysteine endoprotease papain in vitro and its activity was confirmed by assays using a synthetic substrate. Key words: Human cathepsin C, Pichia pastoris, recombinant protein, expression 1. Introduction Cathepsin C (dipeptidyl peptidase I, EC ) is a lysosomal cysteine protease belonging to the papain superfamily (Izumya and Fruton, 1956). Besides its role in lysosomal protein degradation (Coffey and de Duve, 1968), CatC is a key enzyme in the activation of many serine proteinases that