The Use of α-MF Signal Peptide Without Spacer for Producing Insulin Aspart Precursor in Pichia pastoris KM71

Abstract

A spacer peptide (Glu-Ala repeats) connects N-terminal of a polypeptide precursor to the signal peptide of α-mating factor (α-MF) from Saccharomyces cerevisiae. During the polypeptide precursor secretion, the signal peptide will be first cleaved by signal peptidase in the endoplasmic reticulum, followed by Kex2 endopeptidase and finally the spacer peptide is removed by dipeptidyl aminopeptidase in the Golgi apparatus. In the case of insulin precursor, unfortunately, mixture forms of the insulin precursor containing a spacer peptide and the correct insulin precursor is observed in Pichia pastoris. To overcome this problem, a gene encoding insulin Aspart precursor without the spacer peptide has been constructed and its expression has been investigated in this study. The gene of insulin Aspart precursor has been successfully expressed extracellularly in P. pastoris KM71 with molecular mass of 6306.8 Da as determined by LC-ESI-MS. The expression level of insulin Aspart precursor is affected by aeration, cell density and methanol as an inducer. In shake flask production, the insulin Aspart precursor was optimally produced by 3% methanol induction every 24 h for two days, at initial cell density (OD₆₀₀) of 60 and aeration (ratio of culture volume to flask volume) of 1:25. Based on Western blot analysis, there is no intracellular insulin Aspart precursor detected. Therefore, the spacer peptide is not essential for insulin Aspart precursor secretion in P. pastoris and removal of spacer resulted in insulin Aspart precursor homogeneity.