African Journal of
Biotechnology

  • Abbreviation: Afr. J. Biotechnol.
  • Language: English
  • ISSN: 1684-5315
  • DOI: 10.5897/AJB
  • Start Year: 2002
  • Published Articles: 12495

Full Length Research Paper

Intracellular expression of human calcitonin (hCT) gene in the methylotrophic yeast, Pichia pastoris

Ali Salehzadeh1*, Hamideh Ofoghi2, Farzin Roohvand3, Mohammad Reza Aghasadeghi3 and Kazem Parivar1
1Department of Biology, Faculty of Science, Islamic Azad University, Science and Research Branch, Tehran, Iran. 2Iranian Research Organization for Science and Technology, Tehran, Iran. 3Hepatitis and AIDS Department, Pasteur Institute of Iran, Tehran, Iran.
Email: [email protected]

  •  Accepted: 20 May 2011
  •  Published: 03 October 2011

Abstract

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 (muts) strain of P. pastoriswas 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 (muts), Tricine-SDS-PAGE.

Abbreviation

CT, Calcitonin; hCT, human calcitonin; SDS-PAGE, sodium dodecyl sulphate poly acrylamide gel electrophoresis; PMSF, phenylmethylsulfonyl fluoride.