Shangquan Zhu

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 heterologous 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 expression product from the culture medium with an overall recovery of about 80%. After tryptic transpeptidation, human insulin with full receptor binding capacity and biological activity was obtained. In the presence of zinc, the recombinant human insulin could be crystallized in the rhombohedral form.

Protein engineering of insulin: Two novel fast-acting insulins [B16Ala]insulin and [B26Ala]insulin

Blood glucose lowering assay proved that [B16Ala]insulin and [B26Ala]insulin exhibit potency of acute blood glucose lowering in normal pigs, which demonstrates that they are fastacting insulin. Single-chain precursor of [B16Ala]insulin and [B26Ala]insulin is [B16Ala]PIP and [B26Ala]PIP, respectively, which are suitable for gene expression. Secretory expression level of the precursors in methylotrophic yeast Pichia pastoris was quite high, 650 mg/L and 130 mg/L, respectively. In vivo biological assay showed that the two fast-acting insulins have full or nearly full biological activity. So both [B16Ala]insulin and [B26Ala]insulin can be well developed as fast-acting insulin for clinic use.

Insulin and insulin mutants stimulate glucose uptake in rat adipocytes

A simple method to determine thein vitro biological activity of insulin by measuring glucose uptake in the rat adipocytes is presented here. In the presence of insulin, the glucose uptake is 5–6 times more than the basal control. And the uptake of D-[3-3H]-glucose is linear as the logarithm of insulin concentration from 0.2 ώg/L to 1.0 ώg/L. Glucose and 3-O-methyl-glucose inhibit D-[3-3H]-glucose uptake into adipocytes. By this method, thein vitro biological activity of [B2-Lys]-insulin and [B3-Lys]-insulin was measured to be 61.6% and 154% respectively, relative to that of insulin.