Human insulin from a precursor overexpressed in the methylotrophic yeast Pichia pastoris and a simple procedure for purifying the expression product

The methylotrophic yeast Pichia pastoris, which proved successful in produc ing many heterologous proteins, was used to express an insulin precursor. A transformant with a high copy number of the gene integrated into the chrom osome was obtained by the dot-blotting method. In high-density fermentation using a: simple culture medium composed mainly of salt and methanol, the e xpression level reached 1.5 g/L. A simple two-step method was established t o purify the expression product from the culture medium with an overall rec overy of about 80%. After tryptic transpeptidation, human insulin with full receptor binding capacity and biological activity was obtained. In the pre sence of zinc, the recombinant human insulin could be crystallized in the r hombohedral form. (C) 2001 John Wiley & Sons, Inc.