Combinatorial Cdna Library by Complementation of an Auxotrophic Escherichia Coli: Antibodies for Orotate Decarboxylation JEFFREY A

Proc. Natd. Acad. Sci. USA Vol. 91, pp. 8319-8323, August 1994 Biochemistry Selection of catalytic antibodies for a biosynthetic reaction from a combinatorial cDNA library by complementation of an auxotrophic Escherichia coli: Antibodies for orotate decarboxylation JEFFREY A. SMILEY AND STEPHEN J. BENKOVIC* Department of Chemistry, The Pennsylvania State University, University Park, PA 16802 Contributed by Stephen J. Benkovic, May 23, 1994 ABSTRACT Antibodies capable of decarboxylating orotate were sought by immunization with a hapten designed to elicit antibodies with combining sites that resemble the orotate- binding and catalytic portion of the active site of the enzyme OPRTase ODCase orotidine 5'-monophosphate (OMP) decarboxylase (orotidine- 0 '00 \\ 5'-monophosphate carboxy-lyase, EC 4.1.1.23). Active recombinant antibody fragments (Fabs) were selected from a com- HN 'PO4 binatorial cDNA library by complementation of a pyrF strain UMP ofEscherichia cofl and growth ofthe library-expressing cells on pyrimidine-free medium. In this biological screen, a suffi- H " 0 ciently active antibody from the library would decarboxylate Antibody \ Urasi orotate to produce uracil, a pyrimidine source for the aux- Catalysis PRTase otroph, and would provide the cells with a growth advantage compared to cells without an active antibody. Six recombinant AN Fabs yielded identifiable colonies in a screen of 16,000 trans- H formants. To enhance its stability and expression level, one of the six positive fragments was converted into single-chain form. FIG. 1. Pathways for UMP biosynthesis. Upper route involves In this form, the antibody a naturally occurring enzymes orotate phosphoribosyltransferase fragment conferred definite growth (OPRTase) and OMP decarboxylase (ODCase). Mutants lacking one advantage to the auxotroph that was eliminated when the or both of these enzymes could complete a route to UMP with an hapten was included in the medium. The purified single-chain antibody for orotate decarboxylase and the naturally occurring antibody displayed orotate decarboxylase activity in vitro, as enzyme uracil phosphoribosyltransferase (Uracil PRTase). determined by a 14CO2 displacement assay. The specific activity of the antibody is 10-7 times that of naturally occurring tidine-5'-monophosphate carboxy-lyase, EC 4.1.1.23) could OMP decarboxylase, but this antibody-catalyzed rate is esti- synthesize UMP if provided with a replacement enzyme mated to be 108 times the background rate. The results offer the (e.g., a catalytic antibody) with orotate decarboxylase activ- potential to use these methods to obtain catalytic antibodies for ity, since the product, uracil, could be converted to the other biosynthetic reactions as well as to assess the effectiveness nucleotide UMP by the salvage pathway enzyme uracil of the hapten transition state or active site analog in eliciting phosphoribosyltransferase (Fig. 1). antibody catalysts. Our method of selection for active catalytic antibodies described here is to induce an immune response to a hapten One of the potential applications in the development of designed to elicit an antibody with a binding site that may be catalytic antibodies (1, 2) is to create catalysts that can serve capable of orotate decarboxylation; isolate antibody cDNA as replacement enzymes in organisms that lack the enzyme of and assemble a combinatorial heavy- and light-chain phage- interest (3) due either to genetic mutation or to the lack ofthe mid library; infect a pyrimidine auxotrophic E. coli strain metabolic pathway of which the enzyme of interest is a with the antibody library-encoding phage; and evaluate the component. Accompanying the realization ofthis goal would catalytic activity ofantibodies produced in bacterial colonies be the development of methods to select active antibodies, that arise on pyrimidine-free medium. This method may be produced in Escherichia coli or other microorganisms, by generally applicable for selection of antibodies that catalyze utilizing the antibody's ability to catalyze the missing reac- other metabolic reactions. The process of determining the tion in vivo, thus providing the cell with a necessary metab- number of members in a combinatorial library with catalytic olite and a growth advantage on restrictive medium. Of the activity also provides a statistically meaningful assay for many chemical reactions that catalytic antibodies have been evaluation of the effectiveness of transition-state analogues found to accelerate (2), however, few to date involve reac- or other haptens in eliciting catalytic antibodies. tants or products that are found in metabolic pathways. We have attempted to create antibodies capable of the MATERIALS AND METHODS decarboxylation of orotate, an intermediate in the de novo pyrimidine biosynthetic pathway. In the naturally occurring Hapten Design. The design of a hapten to be used for pathway (4), orotate is first converted to the nucleotide generating antibodies capable of orotate decarboxylation is orotidine 5'-monophosphate (OMP), which is then decarbox- based on the mechanistic studies on naturally occurring OMP ylated to yield UMP, the precursor for all other pyrimidine nucleotides. An auxotroph lacking OMP decarboxylase (oro- Abbreviations: OMP, orotidine 5'-monophosphate; Cam, chloram- phenicol; IPTG, isopropyl P-D-thiogalactopyranoside; SCA, single- chain antibody; LcFv, light-chain antibody variable region; HcFv, The publication costs of this article were defrayed in part by page charge heavy-chain antibody variable region; BMP, barbituric acid ribonu- payment. This article must therefore be hereby marked "advertisement" cleotide (6-hydroxy-UMP). in accordance with 18 U.S.C. §1734 solely to indicate this fact. *To whom reprint requests should be addressed. 8319 Downloaded by guest on September 27, 2021 8320 Biochemistry: Smiley and Benkovic Proc. NatL Acad. Sci. USA 91 (1994) decarboxylase. Fig. 2A shows the transition state for decar- by the pelB leader sequence and anchored in the inner boxylation of the zwitterion intermediate. This enzyme ap- membrane by the gIII fusion protein (13). Electroporation- pears to have two major catalytic functions, as first postu- competent JS5 cells (Bio-Rad) were transformed with prod- lated by Beak and Siegel (5): (i) protonation ofthe pyrimidine ucts of the separate heavy- and light-chain ligations, and substrate (6) with a cationic side chain [a lysine, invariant in aliquots of the transformation mixtures were plated on LB all known sequences (7)], leading to the zwitterion interme- agar plus Cam to assess the number of total transformants diate; and (ii) immersion of the charged carboxylate of the and thus the number ofmembers in the individual heavy- and substrate into a hydrophobic portion of the binding site, light-chain libraries. which catalyzes CO2 production by disfavoring the increased The combinatorial library was made by isolation of heavy- electron density on the charged carboxylate. A hapten for chain cDNA from the corresponding plasmid library, and eliciting an antibody binding (and possibly catalytic) site with ligation with restriction-digested light-chain library plasmid. these features should be designed to recruit a cationic proton- Electrocompetent JS5 cells were transformed with products donating amino acid side chain ofthe antibody into proximity of the combinatorial ligation, and the number of total trans- with the C2-C4 ring segment of the substrate, while simul- formants was determined in order to calculate the number of taneously creating a hydrophobic pocket in the active site members in the combinatorial library, as with the individual into which the carboxylate ofthe substrate would be situated. libraries. The remainder of the transformation mixture was We have chosen an N-substituted 2,4-dihydroxyquinoline grown in superbroth (SB; ref. 13) liquid medium (with tetra- as a hapten designed to produce an antibody binding site with cycline and Cam), and plasmid DNA was isolated. For the two catalytic features necessary for orotate decarboxy- production of intact phage, electrocompetent cells were lase activity (Fig. 2B). The conjugated enediolate (C2-C4) of transformed with an amount ofthis plasmid library sufficient this molecule has a pKa value of '-6.5 and is thus anionic at to yield a number of transformants in 10-fold excess of the physiological pH, suitable for inducing a positively charged library size, and the transformation mixture was incubated as residue within the antibody combining site. This charge described except that helper phage VCSM13 (Stratagene) complementarity approach is similar to a hapten design was added with kanamycin for selection (14). After overnight previously used (8). The fused phenyl ring is designed to incubation, cells were removed by centrifugation and phage create a hydrophobic space within the antibody binding site were precipitated by the addition of 2% PEG 8000 and 1.5% in a way similar to that used for obtaining antibodies for NaCl. Phage were stored at 40C and used within 1 week of decarboxylation of carboxybenzisoxazoles (9). N-Carboxy- preparation. propyl-2,4-dihydroxyquinoline (compound 1) was synthe- Infection of Pyrimidine Auxotroph with Antibody-Encing sized by a published method (10) and this hapten was Phage: Biological Screen for Decarboxylase Activity. E. coli conjugated to the carrier protein keyhole limpet hemocyanin strain JFS116 (pyrF, F'lacIQ; ref. 15) was used to attempt for mouse immunization (11). complementation of pyrimidine

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