Yeast expression of a catalytic antibody with chorismate mutase activity

Bowdish, K., Tang, Y., Hicks, J. B., Hilvert, D. (1991) Yeast expression of a catalytic antibody with chorismate mutase activity. Journal of Biological Chemistry, 266 (18). pp. 11901-11908. ISSN 00219258 (ISSN)

URL: http://www.ncbi.nlm.nih.gov/pubmed/2050687

Abstract

The catalytic antibody 1F7 promotes the rearrangement of chorismate into prephenate. We cloned and sequenced the genes encoding this catalyst to determine the origin of the observed rates and specificity. The antibody cDNAs were modified and inserted into inducible expression vectors. Simultaneous intracellular expression of the light and truncated heavy chains in strains of the yeast Saccharomyces cerevisiae lacking natural chorismate mutase resulted in the production of properly folded and assembled Fab antibody. Assembly of the light and heavy immunoglobulin chains is roughly 60-70% efficient in our in vivo system, lagging behind light chain synthesis throughout log and stationary phase. Nevertheless, high intracellular levels of functional Fab antibody (0.1% of total cellular protein) were obtained with an ultra-high copy number plasmid. As yeast-derived 1F7(Fab) catalyzes the chorismate mutase reaction with the same specific activity as antibody isolated from the hybridoma, our expression system now makes possible the application of classical and ''reverse'' genetics to the study and improvement of this first-generation abzyme.

Item Type: Paper
Uncontrolled Keywords: antibody chorismate mutase article gene expression nonhuman priority journal yeast Amino Acid Sequence Antibodies, Fungal Base Sequence Catalysis Cloning, Molecular DNA Electrophoresis, Polyacrylamide Gel Fluorescent Antibody Technique Gene Expression Regulation, Fungal Genes, Fungal Immunoglobulins, Fab Molecular Sequence Data Plasmids Saccharomyces cerevisiae Support, Non-U.S. Gov't Transcription, Genetic
Subjects: organism description > yeast
CSHL Authors:
Communities: CSHL labs > Hicks lab
Depositing User: Matt Covey
Date: 1991
Date Deposited: 09 Aug 2013 20:56
Last Modified: 12 Aug 2013 14:45
Related URLs:
URI: http://repository.cshl.edu/id/eprint/28505

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