Yeast expression of a catalytic antibody with chorismate mutase activity.
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.
Bowdish
K
K
Department of Chemistry, Research Institute of Scripps Clinic, La Jolla, California 92037.
Tang
Y
Y
Hicks
J B
JB
Hilvert
D
D
D000595:Amino Acid Sequence; D000908:Antibodies, Fungal / Q000235:genetics*; D001483:Base Sequence; D002384:Catalysis; D002826:Chorismate Mutase / Q000378:metabolism*; D003001:Cloning, Molecular; D004247:DNA / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D005455:Fluorescent Antibody Technique; D015966:Gene Expression Regulation, Fungal; D005800:Genes, Fungal; D007140:Immunoglobulin Fab Fragments / Q000235:genetics; D008969:Molecular Sequence Data; D010957:Plasmids; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics*; D014158:Transcription, Genetic
D000908:Antibodies, Fungal; D007140:Immunoglobulin Fab Fragments; D004247:DNA; D002826:Chorismate Mutase