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

Abstract

We have established a growth selection requirement for a catalytic antibody with modest chorismate mutase activity. Conversion of (-) chorismate into prephenate is the key step in the biosynthesis of the aromatic amino acids tyrosine and phenylalanine. Strains of the yeast Saccharomyces cerevisiae containing an insertion mutation in the structural gene for the enzyme chorismate mutase (EC 5.4.99.5) require exogenous supplements of these two amino acids for efficient growth. Intracellular expression of the heterologous antibody catalyst in one such strain, identified by random nutagenesis and genetic selection, provides a substantial growth advantage under auxotrophic conditions; complementation was not observed with an unrelated esterolytic antibody. In addition to demonstrating that tailored immunoglobulin catalysts can carry out vital biochemical reactions in vivo, these experiments provide a powerful selection assay for identifying genetic changes within the antibody molecule itself that augment chemical efficiency.Antibodies generated against appropriately designed transition state analogs catalyze a wide variety of chemical transformations (1, 2). Like naturally occurring enzymes, catalytic antibodies achieve substantial rate accelerations with high regio-and stereoselectivity under mild aqueous conditions. As both the mechanism and recognition properties of immunoglobulin catalysts are defined by the structure of the immunizing antigen, this technology provides a potentially general method for preparing tailored enzyme-like molecules on demand for practical applications in chemistry, biology, and medicine. For example, the specificity and biocompatibility of catalytic antibodies suggest that they might be useful for effecting important chemical transformations in vivo. Here we demonstrate the feasibility of this proposition by showing that an antibody with modest chorismate mutase (EC 5.4.99.5) activity (3) can function inside a yeast cell lacking the natural enzyme and confer a growth advantage to its host by virtue of its catalytic activity.MATERIALS AND METHODS Antibody Expression. The genes encoding the catalytic antibody 1F7 were expressed as described (4) using plasmid pABZ260 and the chorismate mutase-deficient Saccharomyces cerevisiae strain YT-4Ca (MATa, aro7::HIS3, leu2, ura3). Plasmid pABZ265, carrying the genes encoding the esterolytic catalytic antibody 6D4 (5, 6), served as a control (see Fig. 2).In Vitro Mutagenesis and Selection. Cells transformed with pABZ260 were subjected to random mutagenesis with ethyl methanesulfonate according to standard procedures (7). Survival after mutagenesis was -20%6. Approximately 5 X 109 mutagenized cells were spread on solid yeast extract/ peptone/dextrose medium, allowed to grow at 300C for 2 days, and replicated onto tyrosine dropout plates containing 2% galactose. Colonies that appeared under selective conditions were picked and plated on phenylalanine dropout plates containing 2% galactose in order to isolate single colonies. Purified mutants ...

Production of a Functional Catalytic Antibody ScFv-NusA Fusion Protein in Bacterial Cytoplasm

“…The peak of product ketone 3 from the analytical RP-C18 column was collected and lyophilized, and the residue taken was up in hexane/isopropanol = 1:1, and analyzed on a chiral-phase column (Chiralpak OD, Daicel, 22 cm ´ 0.45 cm), which allows the separation of the two enantiomeric products (eluent: hexane/isopropanol=7:3; 1 ml/min) as t R (R)-3 = 8. to improve the activity of catalytic antibodies over rounds of mutation and selection. Cytoplasmic expression of functional antibodies is particularly important in that respect as most genetic selection schemes should involve the catalysis of chemical reactions taking place within the cytoplasm, such as the catalysis of key metabolic reactions (11,12) or the liberation of essential growth factors from synthetic precursors. Catalytic antibody 14D9 has been shown to catalyze a variety of processes, such as epoxide and acetal hydrolysis (12,(27)(28)(29)(30).…”

Genetic selection strategies for generating and characterizing catalysts

“…Expression of the 1F7 genes in yeast yielded correctly assembled, functional Fab protein in a = l0-fold higher intracellular concentration than the natural CM (100,101). Since the Fab fragment possesses a 104-fold lower catalytic activity, 1F7 Fab-producing yeast cells which lack the natural enzyme may only exhibit 10-3 of the wildtype activity.…”

“…Since the Fab fragment possesses a 104-fold lower catalytic activity, 1F7 Fab-producing yeast cells which lack the natural enzyme may only exhibit 10-3 of the wildtype activity. This level of enzyme activity is insufficient to relieve the Tyr and Phe requirement of the original CM-deficient yeast strain YT-4Ca (lOl), but provides an obvious growth advantage on selection media to a permissive host strain which was derived from YT-4Ca by random chemical mutagenesis (100).…”

Real and artificial histories

“…Clearly, starting with a population of aptamers that can already bind the substrate greatly increases the chances of finding RNAs with the desired kinase activity. The use of catalytic antibodies to complement auxotrophic microorganisms provides an example of one way in which protein sequence space may be explored 6 • And protocols such as that for in vitro homologous recombination of pools of selected mutant genes -DNA shuffling7 -should provide a means of generating the necessary sequence diversity. Whatever the technology used, the ability to effectively mutagenise and select or screen proteins ( or, indeed, any molecule of interest), on a large scale for defined characteristics will have profound implications for both basic research and medicine.…”