ABSTRACT
Yeast has a rigid cell wall comprising an outer layer of glycoproteins and an internal skeletal layer of glucan; heterologous proteins can be targeted to the glycoprotein layer and become covalently linked to the glucan skeleton. Yeast is a eukaryote that has 'generally regarded as safe' (GRAS) status, and is easy to cultivate, so it seems ideally suited for applications including the manufacture of recyclable, immobilized, biocatalysts, whole-cell vaccines, the presentation of peptide or antibody libraries, and the presentation of adhesion or metal-binding proteins.
Subject(s)
Cell Membrane/metabolism , Recombinant Fusion Proteins/metabolism , Saccharomyces cerevisiae Proteins , Saccharomyces cerevisiae/metabolism , Administration, Oral , Antibodies/genetics , Antibodies/metabolism , Biodegradation, Environmental , Biotechnology/trends , Enzymes, Immobilized , Gene Expression , Mannose-Binding Lectins , Mating Factor , Membrane Proteins/metabolism , Peptides/metabolism , Recombinant Fusion Proteins/genetics , Saccharomyces cerevisiae/genetics , Vaccines/administration & dosage , Vaccines/isolation & purificationABSTRACT
The two major hydrophilic regions of the hepatitis B virus surface antigen (HBsAg) have been expressed in the outer mannoprotein layer of the cell wall of "Bakers Yeast", Saccharomyces cerevisiae, by fusing them between the yeast invertase signal sequence and the yeast alpha-agglutinin carboxyterminal cell wall anchoring sequence. The fusion protein contained most of the preS sequences, including the hepatocyte receptor, and part of the S sequence including the "a" determinant, and was expressed from multiple genomic copies (MIRY) using the constitutive PCK promoter. Immunofluorescence studies showed that the fusion protein was detectable at the cell surface and was stably expressed at a relatively high level. Intraperitoneal immunization of mice revealed a very weak response against the S region, and a high response against yeast itself. It is proposed that increasing the amount of the antigen and reducing the number of native cell wall proteins, might lead to a yeast that is usable as a safe and cheap live oral vaccine.
Subject(s)
Hepatitis B Surface Antigens/immunology , Hepatitis B Vaccines/immunology , Peptide Fragments/immunology , Saccharomyces cerevisiae/genetics , Vaccines, Synthetic/immunology , Administration, Oral , Animals , Hepatitis B Surface Antigens/genetics , Immunization , MiceABSTRACT
The sexual adhesion protein of Saccharomyces cerevisiae MAT alpha cells, alpha-agglutinin, could not be extracted from the cell wall with hot sodium dodecyl sulfate (SDS), but became soluble after digestion of the cell wall with laminarinase. This indicates that it is intimately associated with cell wall glucan. A fusion protein was constructed consisting of the signal sequence of yeast invertase, guar alpha-galactosidase, and the C-terminal half of the alpha-agglutinin. Most of the fusion protein was incorporated in the cell wall. A small amount could be extracted with SDS, but most of it could only be extracted with laminarinase. On the other hand, cells containing a construct consisting of the signal sequence of invertase and alpha-galactosidase released most of the alpha-galactosidase into the medium and all cell wall-associated alpha-galactosidase was released by SDS. Labelling with antibodies showed that the alpha-galactosidase part of the fusion protein was exposed on the surface of the cell wall. The results demonstrate that the C-terminal half of the alpha-agglutinin contains the information needed to incorporate a protein into the cell wall.
