Expression of Diphtheria Toxin Fragment A and Hormone-Toxin Fusion Proteins in Toxin-Resistant Yeast Mutants

• Published in: Proceedings of the National Academy of Sciences - PNAS Vol. 85; no. 22; pp. 8386 - 8390
• Main Authors: Perentesis, John P., Genbauffe, Francis S., Veldman, Sarah A., Galeotti, Cesira L., Livingston, Dennis M., Bodley, James W., Murphy, John R.
• Format: Journal Article
• Language: English
• Published: Washington, DC National Academy of Sciences of the United States of America 01.11.1988; National Acad Sciences
• Subjects: alpha-MSH - genetics; alpha-MSH - isolation & purification; Amino Acid Sequence; Base Sequence; Biochemistry; Biological and medical sciences; Corynebacterium diphtheriae; Diphtheria; Diphtheria Toxin - biosynthesis; Diphtheria Toxin - genetics; Diphtheria Toxin - isolation & purification; DNA; Escherichia coli - genetics; Eukaryotic cells; Fundamental and applied biological sciences. Psychology; Gels; Genes; Genes, Bacterial; Growth, nutrition, metabolism, transports, enzymes. Molecular biology; Interleukins; Microbiology; Molecular Sequence Data; Mutation; Mycology; Peptide Fragments - biosynthesis; Peptide Fragments - genetics; Peptide Fragments - isolation & purification; Plasmids; Recombinant Fusion Proteins - biosynthesis; Recombinant Fusion Proteins - isolation & purification; Recombinant Proteins - isolation & purification; Saccharomyces cerevisiae; Saccharomyces cerevisiae - genetics; Secretion; Toxins; Yeasts; Infection; Hormone; Intermolecular melting; Toxin; Phenotype; Yeast; Bacteriosis; Mutation; Diphtheria; Genetic transfer
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.85.22.8386

Mutants of the eukaryote Saccharomyces cerevisiae, previously selected for resistance to diphtheria toxin, were investigated for their suitability as hosts for the expression of tox-related proteins. The structural gene for the toxin, encoding the fragment A catalytic domain, was modified for efficient intracellular expression in eukaryotes and placed downstream of the yeast GAL1 promoter element in a plasmid. Transformed mutant yeast grown in galactose, which induces that promoter, were viable and contained active fragment A. In contrast, sensitive, wild-type cells harboring this plasmid grew normally under repressing conditions but were killed when the GAL1 promoter was induced. Additional constructions were also prepared that included sequences encoding either the lymphocyte growth factor interleukin 2 or α -melanocyte-stimulating hormone along with the lipid-associating domains of fragment B and the leader peptide of the Kluyveromyces lactis killer toxin. Resistant mutant strains transformed with these plasmids efficiently expressed and secreted the expected chimeric toxins.