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

摘要

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).