COMMUNICATION TO THE EDITOR 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 producing many heterolo- gous proteins, was used to express an insulin precursor. A transformant with a high copy number of the gene integrated into the chromosome was obtained by the dot-blotting method. In high-density fermentation using a simple culture medium composed mainly of salt and methanol, the expression level reached 1.5 g/L. A simple two-step method was established to purify the expres- sion product from the culture medium with an overall recovery of about 80%. After tryptic transpeptidation, hu- man insulin with full receptor binding capacity and bio- logical activity was obtained. In the presence of zinc, the recombinant human insulin could be crystallized in the rhombohedral form. © 2001 John Wiley & Sons, Inc. Biotech- nol Bioeng 73: 74-79, 2001.