African Journal of Biotechnology Vol. 10(59), pp. 12691-12696, 3 October, 2011 Available online at http://www.academicjournals.org/AJB DOI: 10.5897/AJB10.2359 ISSN 1684–5315 © 2011 Academic Journals Full Length Research Paper Intracellular expression of human calcitonin (hCT) gene in the methylotrophic yeast, Pichia pastoris Ali Salehzadeh 1 *, Hamideh Ofoghi 2 , Farzin Roohvand 3 , Mohammad Reza Aghasadeghi 3 and Kazem Parivar 1 1 Department of Biology, Faculty of Science, Islamic Azad University, Science and Research Branch, Tehran, Iran. 2 Iranian Research Organization for Science and Technology, Tehran, Iran. 3 Hepatitis and AIDS Department, Pasteur Institute of Iran, Tehran, Iran. Accepted 20 May, 2011 This study utilized the Pichia pastoris expression system for expression of the synthetic human calcitonin (hCT) gene, a small peptide hormone secreted by the thyroid gland in mammals and ultimobranchial glands in lower vertebrate. The P. pastoris vector (pPICZB) contains the alcohol oxidase gene promoter (AOX1), which under the induction of methanol allows for the expression of heterologous protein gene inserted downstream in the vector. KM71H (mut s ) strain of P. pastoris was used as the host cell. Molecular analysis, including polymerase chain reaction (PCR), sequencing, restriction enzyme analysis and survival of P. pastoris to increase concentration of zeocin antibiotic showed that human calcitonin gene was successfully integrated into the P. pastoris genome. The expected peptide which had an apparent molecular mass of 5.5 kDa was detected by Tricine-SDS-PAGE analysis and confirmed by enzyme-linked immunosorbent assay (ELISA). Key words: Pichia pastoris, human calcitonin, KM71H (mut s ), Tricine-SDS-PAGE. INTRODUCTION Calcitonin (CT) is a peptide hormone produced by specialized C-parafollicular cells of the thyroid glands in mammals or by cells of the ultimobranchial glands in fish and reptiles. CT plays an important role in regulating phosphorus and calcium metabolism, decreasing blood calcium concentrations and inhibiting bone resorption. Natural CT and synthesized analog are widely used in clinical practice for the treatment of postmenopausal osteoporosis, Paget’s disease of bone, bone pain, spinal stenosis, acute pancreatitis and gastric ulcer (Li et al., 2009). Following the increase of the proportion of the elderly people in the world, osteoporosis has become a *Corresponding author. E- mail: salehzadehmb@yahoo.com. Tel: +989126932196. Abbreviations: CT, Calcitonin; hCT, human calcitonin; SDS- PAGE, sodium dodecyl sulphate poly acrylamide gel electrophoresis; PMSF, phenylmethylsulfonyl fluoride. major threat to the public health due to its high morbidity and mortality (Lim et al., 2004). Low bone mass and deterioration of bone micro architecture are the major characteristics of osteoporosis, which results in increased bone brittleness and thus is associated with an increased risk of fracture. CT is one of the effective and safe agents for the treatment of osteoporosis (Munoz-Torres et al., 2004). Gills of salmon and pig thyroid glands are the main source of CT that is used in clinical practice (Tanko et al., 2004). However, these heterologous products are short of resources and thus expensive. CT activity is not species-specific which make it possible to use animal CT (porcine, salmon and eel) for treatment of human patients. However, due to immunological reactions, the prolonged application of animal CT leads to a gradual decrease or loss of activity. That is why the long term treatment of human patients with CT requires homologous human calcitonin (hCT)(Azria 1989). Thus, genetic engineering techniques with hCT gene as the target gene may provide solutions to the earlier- mentioned problem. In this study, we described the