title: A new means of inducibly inactivating a cellular protein.
abstract: This paper presents a general means of eliminating the function of a single protein without relying on genetic alterations in its structure or level of synthesis. The strategy is based on the inducible cellular expression of neutralizing antibody to inactivate the protein selectively. The feasibility of this approach is illustrated by using alcohol dehydrogenase I (ADH I) in Saccharomyces cerevisiae as a model. Heavy- and light-chain cDNAs were isolated from a hybridoma secreting an antibody which neutralizes yeast ADH I. The cDNAs were characterized with respect to their length and identity, their signal sequences were removed, and synthetic translation initiation codons were joined to them. These truncated sequences were then inserted into an inducible expression vector and shown to be expressed as stable heavy and light chains, which assemble and bind antigen. The sequences were introduced into yeast mutants containing different levels of ADH activity, and evidence is provided that the antibodies produce limited neutralization of enzyme activity in vivo. In principle, the approach can be used for any cell type in which functional antibody can be inducibly expressed.
mesh_terms: D000426:Alcohol Dehydrogenase / Q000276:immunology*; D001483:Base Sequence; D005803:Genes, Immunoglobulin; D005813:Genes, Synthetic; D005822:Genetic Vectors; D007074:Immunoglobulin G / Q000096:biosynthesis* / Q000276:immunology; D007143:Immunoglobulin Heavy Chains / Q000235:genetics; D007145:Immunoglobulin kappa-Chains / Q000235:genetics; D008969:Molecular Sequence Data; D010442:Peptide Chain Initiation, Translational; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000276:immunology*
chemical_list: D007074:Immunoglobulin G; D007143:Immunoglobulin Heavy Chains; D007145:Immunoglobulin kappa-Chains; D000426:Alcohol Dehydrogenase