Question

High level expression and efficient assembly of hepatitis b surface antigen in the methylotrophic yeast, pichia pastoris

Answer 1

The methylotrophic industrial yeast, Pi–chia pastoris, has been developed as a host system for the large–scale production of heterologous proteins. As an example of the systems, the synthesis of hepatitis B surface antigen (HBsAg) is described. Expression of the HBsAg gene is controlled by a methanol–regulated promoter derived from the primary alcohol oxidase gene, AOX1, of P. pastoris. The highest level of HBsAg is observed in a novel mutant strain of P. pastoris in which the AOX1 structural gene is deleted and replaced by a construction composed of the AOX1 promoter–HBsAg gene expression cassette. When grown on methanol, 2–3% of the soluble protein in this strain is HBsAg. characterization of the recombinant HBsAg indicates that virtually all of the P. Pastoris–Synthesized HBsAg exists in a form which is similar to human serum–derived HBsAg 22 nm particles. Scale–up of the HBsAg production process from shake–flask to high cell–density fermentor cultures is described as well. A single culture of the P. Pastoris HBsAg expression strain in a volume of 240 Liters and at a density of 60 grams/Liter dry weight of cells produced 0.4 grams/Liter, or a total of 90 grams of HBsAg 22 nm particles, an amount sufficient for approximately 9 million doses of vaccine.