Expression of hepatitis B surface antigen gene in yeast (recombinant DNA/serology/gene engineering/vaccine/acid phosphatase promoter)

The DNA sequence coding for hepatitis B virus surface antigen (HBsAg) was placed under control of the repres- sible acid phosphatase promoter of the yeast Saccharomyces cere- visiae in a plasmid capable of autonomous replication in both yeast and Escherichia coli. Yeast transformed by this plasmid synthe- sized up to 5 x 105 molecules per cell of immunologically active HBsAg polypeptide in phosphate-free medium. The HBsAg poly- peptides produced in the yeast cells were assembled into 20- to 22- nm spherical or oval particles and were immunogenic. Hepatitis B virus (HBV) causes serious human liver disease, including hepatoma. Infection by this virus is a worldwide health problem, and a considerable number of people, partic- ularly in Southeast Asia, the Middle East, and some areas of Africa, suffer from transient or chronic HBV infection. The in- fectious agent has been identified as a 40- to 50-nm spherical particle, called the Dane particle, that is detected in patients' blood. The Dane particle contains a 3.2-kilobase (kb) circular DNA that has a single-stranded gap (1, 2). Intensive efforts have been made to understand the struc- tural and behaviorial characteristics of this virus in order to con- trol the disease and to produce vaccines. However, such efforts have been hampered seriously by the fact that HBV replicates only in human and chimpanzee livers. The viral genome has been converted to double-stranded form by filling-in and has been cloned and propagated in Esch- erichia coli (3-6). Analyses of the cloned HBV genome have brought new insights into the structure and function of this viral genome. At the same time, it was expected that vaccines might be produced in E. coli cells by allowing expression of the cloned surface antigen (HBsAg) gene. Despite many efforts, however, production of vaccines in E. coli has not succeeded because, in E. coli cells, the HBsAg gene product seems to be either un- stable or to cause effects deleterious to the host, or both. In the present study, we report a yeast system that allows expression of the HBsAg gene. One of the yeast acid phosphatases (APase; orthophosphoric- monoester phosphohydrolase (acid optimum), EC 3.1.3.2) is an exocellular 60-kilodalton polypeptide designated P-60; the expression of the gene for P-60 is controlled by the level of in- organic phosphate (7-12). We took this gene, freed its promoter from the coding sequence of the polypeptide, and joined the promoter with the HBsAg gene in such a way that free, non- fusion polypeptides of HBsAg were made. The promoter- HBsAg gene complex was inserted into a shuttle vector that replicates in both yeast and E. coli and then was used to trans- form yeast. The transformants produce a large quantity of HBsAg polypeptide in low Pi medium, and the HBsAg is as- sembled into spherical or oval immunogenic particles.