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. There is ample precedent from microinjection experiments for the ability of antibodies to cause phenotypic effects in vivo (see, e.g., references 9 and 26). In one such experiment, antibodies were shown to be able to react with antigen in the cytoplasm for at least 6 h (30). Other methods of eliminating the activity of specific proteins, including one based on the use of antisense RNA, have recently been developed (15) or proposed (12), although the extent of their utility is not yet clear.As a model system in which to test the feasibility of this approach, the yeast enzyme alcohol dehydrogenase I (ADH I) was chosen because of the extent of its physical (19) and genetic (6, 31) characterization, its cytoplasmic location, the availability of a convenient in vitro assay for its activity, and, above all, the ability to select both for and against its activity. Three monoclonal antibodies which potently neutralize ADH activity in a yeast extract and show no detectable cross-reactivity with other cell components are described in the accompanying paper (5).The present article describes the isolation of heavy-and light-chain cDNA clones encoding one of these antibodies, the removal of their signal sequence coding regions, and the addition of synthetic translation initiation codons to their 5' ends. These truncated sequences are then inserted into a single expression vector in such a manner that both are transcribed coordinately under inducible control in the yeast Saccharomyces cerevisiae. Stable heavy-and light-chain polypeptides are synthesized, assemble, and bind antigen. Finally, evidence is presented that limited in vivo neutralization occurs upon induction of antibody synthesis. MATERIALS AND METHODSConstruction and screening of a hybridoma cDNA library. RNA was extracted from hybridoma cells by extraction in guanidine hydrochloride, and poly(A)+ RN...

Potent inhibition of human immunodeficiency virus type 1 replication by an intracellular anti-Rev single-chain antibody.

“…Modified antibodies can be stably expressed intracellularly, within the nucleus and/or the cytoplasm of eukaryotic cells, and have been used to inactivate specific cellular gene products (11,12). In some initial studies, the hydrophobic leader sequences of these molecules were inactivated to allow intracellular cytoplasmic expression (12).…”

In vivo catalysis of a metabolically essential reaction by an antibody.

“…Cells transformed with pABZ260 were subjected to random mutagenesis with ethyl methanesulfonate according to standard procedures (7). Sur- (10) demonstrated a number of years ago that functional antibody molecules can be expressed cytoplasmically in yeast to alter phenotype. An engineered strain of S. cerevisiae containing an antibody directed against yeast alcohol dehydrogenase was shown to exhibit increased growth on allyl alcohol due to limited neutralization of the target enzyme by the immunoglobulin.…”

Intrabodies as Neuroprotective Therapeutics

“…Critically, they lack the potentially inflammatory Fc region. Intrabodies were first reported in 1988 [1], and they have been studied extensively as potential therapeutics for infectious diseases [2][3][4][5] and cancer [6][7][8]. Our work and that of others has recently exploited the specificity and affinity characteristics of intrabodies to combat neurodegenerative diseases that share the cellular and molecular features of protein misfolding and aggregation [9][10][11][12][13].…”