Carboxylic acid reductase polypeptide, nucleotide sequence encoding same and methods of use Number:7,056,714 from the United States Patent and Trademark Office (PTO) owispatent The invention provides the nucleotide sequence and amino acid sequence for the enzyme carboxylic acid reductase isolated from bacteria. Expression cassettes, vectors, transformed cells, and variants are also provided as methods for use of recombinant biocatalytic reagents in production of synthetic, aromatic, aliphatic and alicyclic aldehydes and alcohols.<H polypeptide, nucleotide, Carboxylic, acid, 7056714.html, Patent, pto, 7056714

Title: Carboxylic acid reductase polypeptide, nucleotide sequence encoding same and methods of use

Abstract: The invention provides the nucleotide sequence and amino acid sequence for the enzyme carboxylic acid reductase isolated from bacteria. Expression cassettes, vectors, transformed cells, and variants are also provided as methods for use of recombinant biocatalytic reagents in production of synthetic, aromatic, aliphatic and alicyclic aldehydes and alcohols.

Patent Number: 7,056,714 Issued on 06/06/2006 to Rosazza, et al.

Inventors: Rosazza; John P. (Iowa City, IA); Fotheringham; Ian (Edinburgh, GB); Li; Tao (Union, NJ); Daniels; Lacy (Iowa City, IA); He; Aimin (Iowa City, IA)
Assignee: University of Iowa Research Foundation, Inc. (Iowa City, IA)

Appl. No.: 386329

Filed: March 11, 2003

Current U.S. Class: 435/189 ; 435/25; 435/252.3; 435/252.33; 435/320.1; 435/4; 435/440; 435/6; 435/69.1; 435/71.1; 536/23.2; 536/23.7

Current International Class: C12N 9/02 (20060101); C07H 21/04 (20060101); C12N 1/20 (20060101); C12N 15/00 (20060101); C12P 21/04 (20060101); C12Q 1/00 (20060101); C12Q 1/26 (20060101)

Field of Search: 435/189,252.3,320.1,252.33,69.1,71.1,4,6,440,25 536/23.2,23.7,23.5

References Cited [Referenced By]

U.S. Patent Documents


5795759	August 1998	Rosazza et al.
6261814	July 2001	Rosazza et al.

Other References

Kita et al. Cloning of the aldehyde reductase gene from a red yeast, Sporobolomyces salmonicolor, and characterization of the gene and its product. Appl Environ Microbiol. Jul. 1996;62(7):2303-10. cited by examin- er.

Primary Examiner: Achutamurthy; Ponnathapu
Assistant Examiner: Pak; Yong D.
Attorney, Agent or Firm: McKee, Voorhees & Sease, P.L.C.

Claims

What is claimed is:

1. An isolated polynucleotide encoding a carboxylic acid reductase, said polynucleotide selected from the group consisting of: (a) a polynucleotide having at least 95% nucleotide sequence identity to SEQ ID NO:1 encoding a polypeptide having carboxylic acid reductase activity; (b) a polynucleotide comprising SEQ ID NO:1 encoding a polypeptide having carboxylic acid reductase activity; and (c) a polynucleotide which is complementary to the full length of the polynucleotide of (a) or (b).

2. A recombinant expression cassette comprising a polynucleotide selected from the group consisting of: (a) a polynucleotide having at least 95% nucleotide sequence identity to SEQ ID NO:1 encoding a polypeptide having carboxylic acid reductase activity; (b) a polynucleotide comprising SEQ ID NO:1 encoding a polypeptide having carboxylic acid reductase activity; and (c) a polynucleotide which is complementary to the full length of the polynucleotide of (a) or (b).

3. A vector comprising a recombinant expression cassette comprising a polynucleotide selected from the group consisting of: (a) a polynucleotide having at least 95% nucleotide sequence identity to SEQ ID NO:1 encoding a polypeptide having carboxylic acid reductase activity; (b) a polynucleotide comprising SEQ ID NO:1 encoding a polypeptide having carboxylic acid reductase activity; and (c) a polynucleotide which is complementary to the full length of the polynucleotide of (a) or (b).

4. An isolated host cell transformed with a recombinant expression cassette which comprises a polynucleotide selected from the group consisting of: (a) a polynucleotide having at least 95% nucleotide sequence identity to SEQ ID NO:1 encoding a polypeptide having carboxylic acid reductase activity; (b) a polynucleotide comprising SEQ ID NO:1 encoding a polypeptide having carboxylic acid reductase activity; and (c) a polynucleotide which is complementary to the full length of the polynucleotide of (a) or (b).

5. The host cell of claim 4 wherein the cell is a bacterial cell.

6. The host cell of claim 5 wherein the cell is an E. Coli cell.

7. An isolated bacterial cell transformed with a polynucleotide selected from the group consisting of: (a) a polynucleotide having at least 95% nucleotide sequence identity to SEQ ID NO:1 encoding a polypeptide having carboxylic acid reductase activity; (b) a polynucleotide comprising SEQ ID NO:1 encoding a polypeptide having carboxylic acid reductase activity; and (c) a polynucleotide which is complementary to the full length of the polynucleotide of (a) or (b).

8. A method of making a carboxylic acid reductase enzyme comprising the steps of: a) expressing a polynucleotide in a bacterial host cell transformed with said polynucleotide, wherein the polynucleotide is selected from the group consisting of: i) a polynucleotide having at least 95% nucleotide sequence identity to SEQ ID NO:1 encoding a polypeptide having carboxylic acid reductase activity; and ii) a polynucleotide comprising SEQ ID NO:1 encoding a polypeptide having carboxylic acid reductase activity; b) culturing said bacterial cell under cell growth conditions; so that carboxylic acid reductase is produced and c) harvesting said carboxylic acid reductase.

Description

BACKGROUND OF THE INVENTION

Microorganism-produced enzymes are widely used as a class of biocatalytic reagents in production of synthetic, aromatic, aliphatic and alicyclic aldehydes and alcohols are useful chemical intermediates in chemical, agrochemical, pharmaceutical and food industries. These enzymes are useful in a wide variety of reactions including, e.g., oxidations, reductions, hydrolyses, and carbon--carbon bond ligations.

Biocatalysts are valued for their intrinsic abilities to bind organic substrates and to catalyze highly specific and selective reactions under the mildest of reaction conditions. These selectivities and specificities are realized because of highly rigid interactions occurring between the enzyme active site and the substrate molecule. Biocatalytic reactions are particularly useful when they may be used to overcome difficulties encountered in catalysis achieved by the use of traditional chemical approaches.

Carboxylic acid reductases are complex, multicomponent enzyme systems, requiring the initial activation of carboxylic acids via formation of AMP and often coenzyme A intermediates (see, e.g., Hempel et al., Protein Sci. 2:1890 1900 (1993). Chemical methods for carboxylic acid reductions are generally poor usually requiring prior derivatization and product deblocking with multifunctional reactants.

An enzymatic reaction offers significant advantages over existing methods used in chemical reductions of carboxylic acids, or their derivatives. Unlike many substrates subjected to biocatalytic reactions, carboxylic acids are generally water soluble, rendering them of potentially broad application to this class of enzyme. The carboxylic acid reduction reaction appears to bear the usual desirable features of functional group specificity. It also functions well under mild reaction conditions and produces a high yield of product. The reduction of the activated carboxylic acid intermediate occurs step-wise to give aldehyde products (Gross et al., Eur. J. Biochem. 8:413 419; 420 425 (1969); Gross, Eur. J. Biochem. 31:585 592 (1972)).

The reduction of carboxylic acids by microorganisms is a relatively new biocatalytic reaction that has not yet been widely examined or exploited. Jezo and Zemek reported the reduction of aromatic acids to their corresponding benzaldehyde derivatives by Actinomycetes in Chem. Papers 40(2):279 281 (1986). Kato et al. reported the reduction of benzoate to benzyl alcohol by Nocardia asteroides JCM 3016 (Agric. Biol. Chem. 52(7):1885 1886 (1988)), and Tsuda et al. described the reduction of 2-aryloxyacetic acids (Agric. Biol. Chem. 48(5): 1373 1374 (1984)) and arylpropionates (Chem. Pharm. Bull. 33(11):4657 4661 (1985)) by species of Glomerella and Gloeosporium. Microbial reductions of aromatic carboxylic acids, typically to their corresponding alcohols, have also been observed with whole cell biotransformations by Clostridium thermoaceticum (White et al., Eur. J. Biochem. 184:89 96 (1989)), and by Neurospora (Bachman et al., Arch. Biochem. Biophys. 91:326 (1960)). More recently, carboxylic acid reduction reactions have reportedly been catalyzed by whole cell preparations of Aspergillus niger, Corynespora melonis and Coriolus (Arfmann et al., Z. Naturforsch 48c:52 57 (1993); cf., Raman et al., J. Bacterial 84:1340 1341 (1962)), and by Nocardia asteriodes (Chen and Rosazza, Appl. Environ. Microbiol. 60(4):1292 1296 (1994)).

Biocatalytic reductions of carboxylic acids are attractive to traditional chemical catalysis because the substrates are water soluble, blocking chemistry is not necessary, reductions are enantioselective (7), and the scope of the reaction is very broad (23, 32).

Aldehyde oxidoreductases are also known as carboxylic acid reductases (CAR), require ATP, Mg.sup.2+, and NADPH as cofactors during carboxylic acid reduction (15, 16, 20, 23). The reduction reaction is a stepwise process involving initial binding of both ATP and the carboxylic acid to the enzyme, to form mixed 5'-adenylic acid-carbonyl anhydride intermediates (8, 14, 24, 26, 40) that are subsequently reduced by hydride delivery from NADPH to form the aldehyde product (15, 24).

Aromatic carboxylic acid reductases have been purified to homogeneity only from Neurospora (16) and Nocardia (20, 23). Although details of N- and internal amino acid sequences have been reported for the Nocardia asteriodes enzyme (23), complete gene sequences for these or any other carboxylic acid reductases are unknown.

It is an object of the present invention to provide a purified and isolated bacterial carboxylic acid reductase (CAR) gene and the protein encoded thereby.

It is yet another object of the invention to provide homologous nucleotide sequences and/or amino acid sequences which encode CAR.

It is yet another object of the invention to provide recombinant DNA using expression constructs, vectors, and recombinant cells using the sequences of the invention for production of recombinant CAR.

It is yet another object of the invention to provide for large scale production of and recovery of recombinant CAR, for use in production of synthetic, aromatic, aliphatic and alicyclic aldehydes and alcohols.

It is yet another embodiment of the invention to provide methods of synthesis of chemical compounds such as those for biocatalytically reducing a carboxylic acid, or a derivative thereof, to its corresponding aldehyde product(s), to provide a method of biocatalytically reducing a carboxylic acid, or a derivative thereof, to its corresponding intermediary by-product(s), as exemplified by acyl-AMP analogs, or to provide a method of biocatalytically reducing vanillic acid, or a precursor or derivative thereof, to vanillin, all using recombinant CAR as described the invention disclosed herein.

Additional objects, advantages and novel features of the invention will be set forth in part in the description which follows, and in part will become apparent to those skilled in the art on examination of the following, or may be learned by practice of the invention.

BRIEF SUMMARY OF THE INVENTION

The present invention provides polynucleotides, related polypeptides and all conservatively modified variants of purified and isolated CAR. The nucleotide sequence of CAR comprises the sequence found in SEQ ID NO: 1, 3, and 5. Sequences 3 and 5 provide examples of conservatively modified polynucleotides of SEQ ID NO: 1 and sequences 7, and 9, 11, are examples of sequences with 80, 90, and 95% sequence identity to SEQ ID NO:1 as also described herein.

Therefore, in one aspect, the present invention relates to an isolated nucleic acid comprising an isolated polynucleotide sequence encoding a CAR enzyme. In a further aspect, the present invention includes a nucleic acid selected from: (a) an isolated polynucleotide encoding a polypeptide of the present invention; (b) a polynucleotide having at least 80%, 90% or 95% identity to a polynucleotide of the present invention; (c) a polynucleotide comprising at least 25 nucleotides in length which hybridizes under high stringency conditions to a polynucleotide of the present invention; (d) a polynucleotide comprising a polynucleotide of the present invention; and (e) a polynucleotide which is complementary to the polynucleotide of (a) to (d).

In another aspect, the present invention relates to a recombinant expression cassette comprising a nucleic acid as described, supra. Additionally, the present invention relates to a vector containing the recombinant expression cassette. Further, the vector containing the recombinant expression cassette can facilitate the transcription and translation of the nucleic acid in a host cell. The present invention also relates to host cells able to express the polynucleotide of the present invention. A number of host cells could be used, such as but not limited to, microbial, mammalian, plant, or insect. In a preferred embodiment the host cell is a bacterial cell. In a more preferred embodiment the bacterial host cell is E. Coli. Thus the invention is also directed to transgenic cells, containing the nucleic acids of the present invention as well as cells, strains and lines derived therefrom.

This invention also provides an isolated polypeptide comprising (a) a polypeptide comprising at least 80%, 90% or 95% sequence identity to a polypeptide of the present invention (SEQ ID NO:2); (b) a polypeptide encoded by a nucleic acid of the present invention; and (c) a polypeptide comprising CAR activity and modeled and designed after SEQ ID NO:1.

Another embodiment of the subject invention comprises a methods for biocatalytically reducing a carboxylic acid, or a derivative thereof, to its corresponding aldehyde product(s), to provide a method of biocatalytically reducing a carboxylic acid, or a derivative thereof, to its corresponding intermediary by-product(s), as exemplified by acyl-AMP analogs, or to provide a method of biocatalytically reducing vanillic acid, or a precursor or derivative thereof, to vanillin, all using recombinant cells, extracts, CAR protein purified therefrom or derivatives and modifications of this CAR protein.

Yet another embodiment of the invention comprises a method of making a polypeptide of a recombinant gene comprising: a) providing a population of these host cells; and b) growing the population of cells under conditions whereby the polypeptide encoded by the coding sequence of the expression cassette is expressed; c) isolating the resulting polypeptide.

A number of expression systems using the said host cells could be used, such as but not limited to, microbial, mammalian, plant, or insect.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is an alignment of the deduced amino acid sequence of Nocardia CAR with a representative sample of putative homologous molecules from other organisms. Identical amino acids are highlighted in black, and similar amino acids are highlighted in gray. The Clustal W program was used to align the above sequences, and Boxshade (0.7 setting) was used to determine the degree of residue shading. The corresponding nucleotide sequence encoding Nocardia CAR has been deposited in the GenBank/EMBL database. Accession nos. for the other protein sequences above are: MtfadD, M. tuberculosis (Z77724), Mlacl, M. leprae (NP.sub.--301424), Msmeg, M. smegmatis (Contig 3313), MBCG, M. bovis BCG (unnamed hypothetical protein at bases 2,885,319 2,888,822).

FIGS. 2a and b are SDS-PAGE (a) and Western blot (b) analysis of Nocardia CAR expression in E. coli carrying pHAT10 based vectors. Samples taken from the lysates of E. coli cells carrying different vectors were separated in duplicate by 10% SDS-PAGE and either stained with 0.1% Coomassie blue R-250 (A) or subjected to Western blotting using a HAT-specific antibody (B). Lane assignments for panels A and B: 1, molecular weight markers: myosin (209 kDa), beta-galactosidase (124 kDa), BSA (80 kDa), ovalbumin (49.1 kDa), carbonic anhydrase (34.8 kDa), soybean trypsin inhibitor (21.5 kDa) and lysozyme (20.6 kDa), aprotinin (7.1 kDa); 2, E. coli cells BL21-CodonPlus.RTM. (DE3)-RP carrying pHAT-DHFR; 3, E. coli BL21(DE3) cells carrying pHAT-305; 4, E. coli BL21-CodonPlus.RTM. (DE3)-RP cells carrying pHAT-305 (uninduced); 5, purified HAT-CAR; 6, E. coli CodonPlus.RTM. (DE3)-RP cells carrying pHAT10.

FIG. 3 depicts the alpha-Aminoadipate reductase motifs that were described by Casqueiro at al. and Hijarrubia et al. that are present in Car. Red letters indicate identical amino acids and blue letters indicate similar amino acids. Bold letters are matches within the motif.

FIG. 4 depicts the location of motifs within Car

FIG. 5 depicts the location of motifs within FadD9.

FIG. 6 depicts the location of motifs in Aar: yeast AAR.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Unless mentioned otherwise, the techniques employed or contemplated herein are standard methodologies well known to one of ordinary skill in the art. The materials, methods and examples are illustrative only and not limiting. The following is presented by way of illustration and is not intended to limit the scope of the invention.

The practice of the present invention will employ, unless otherwise indicated, conventional techniques of botany, microbiology, tissue culture, molecular biology, chemistry, biochemistry and recombinant DNA technology, which are within the skill of the art. Such techniques are explained fully in the literature. See, e.g., The Microbial World, (1986) 5th Ed., Prentice-Hall; O. D. Dhringra and J. B. Sinclair, Basic Plant Pathology Methods, (1985) CRC Press; Maniatis, Fritsch & Sambrook, Molecular Cloning: A Laboratory Manual (1982); DNA Cloning, Vols. I and II (D. N. Glover ed. 1985); Oligonucleotide Synthesis (M. J. Gait ed. 1984); Nucleic Acid Hybridization (B. D. Hames & S. J. Higgins eds. 1984); and the series Methods in Enzymology (S. Colowick and N. Kaplan, eds., Academic Press, Inc.).

Units, prefixes, and symbols may be denoted in their SI accepted form. Unless otherwise indicated, nucleic acids are written left to right in 5' to 3' orientation; amino acid sequences are written left to right in amino to carboxy orientation, respectively. Numeric ranges are inclusive of the numbers defining the range. Amino acids may be referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Nucleotides, likewise, may be referred to by their commonly accepted single-letter codes. The terms defined below are more fully defined by reference to the specification as a whole.

In describing the present invention, the following terms will be employed, and are intended to be defined as indicated below.

By "amplified" is meant the construction of multiple copies of a nucleic acid sequence or multiple copies complementary to the nucleic acid sequence using at least one of the nucleic acid sequences as a template. Amplification systems include the polymerase chain reaction (PCR) system, ligase chain reaction (LCR) system, nucleic acid sequence based amplification (NASBA, Cangene, Mississauga, Ontario), Q-Beta Replicase systems, transcription-based amplification system (TAS), and strand displacement amplification (SDA). See, e.g., Diagnostic Molecular Microbiology: Principles and Applications, D. H. Persing et al., Ed., American Society for Microbiology, Washington, D.C. (1993). The product of amplification is termed an amplicon.

The term "conservatively modified variants" applies to both amino acid and nucleic acid sequences. With respect to particular nucleic acid sequences, conservatively modified variants refer to those nucleic acids that encode identical or conservatively modified variants of the amino acid sequences. Because of the degeneracy of the genetic code, a large number of functionally identical nucleic acids encode any given protein. For instance, the codons GCA, GCC, GCG and GCU all encode the amino acid alanine. Thus, at every position where an alanine is specified by a codon, the codon can be altered to any of the corresponding codons described without altering the encoded polypeptide. Such nucleic acid variations are "silent variations" and represent one species of conservatively modified variation. Every nucleic acid sequence herein that encodes a polypeptide also describes every possible silent variation of the nucleic acid. One of ordinary skill will recognize that each codon in a nucleic acid (except AUG, which is ordinarily the only codon for methionine, one exception is Micrococcus rubens, for which GTG is the methionine codon (Ishizuka, et al., J. Gen'l Microbiol, 139:425 432 (1993)) can be modified to yield a functionally identical molecule. Accordingly, each nucleic acid disclosed herein also includes each silent variation of the nucleic acid, which encodes a polypeptide of the present invention, it is implicit in each described polypeptide sequence and incorporated herein by reference. Examples of conservatively modified variants with silent mutations are SEQ ID NO:37 (where some gca codons have been replaced with gcg condons both of which code for Alanine) and 38 (where a tca codon has been replaced with an agt codon both of which code for serine).

As to amino acid sequences, one of skill will recognize that individual substitutions, deletions or additions to a nucleic acid, peptide, polypeptide, or protein sequence which alters, adds or deletes a single amino acid or a small percentage of amino acids in the encoded sequence is a "conservatively modified variant" when the alteration results in the substitution of an amino acid with a chemically similar amino acid. Thus, any number of amino acid residues selected from the group of integers consisting of from 1 to 15 can be so altered. Thus, for example, 1, 2, 3, 4, 5, 7, or 10 alterations can be made. Conservatively modified variants typically provide similar biological activity as the unmodified polypeptide sequence from which they are derived. For example, substrate specificity, enzyme activity, or ligand/receptor binding is generally at least 80%, or 95%, preferably 80 95% of the native protein for it's native substrate. Conservative substitution tables providing functionally similar amino acids are well known in the art. Sequence ID no 39 is a protein sequence with a conservative substitution of A for S.

The following six groups each contain amino acids that are conservative substitutions for one another: 1) Alanine (A), Serine (S), Threonine (T); 2) Aspartic acid (D), Glutamic acid (E); 3) Asparagine (N), Glutamine (Q); 4) Arginine (R), Lysine (K); 5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); and 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W). See also, Creighton (1984) Proteins W.H. Freeman and Company. Examples of proteins with conservatively modified variants are SEQ ID NO:______.

By "encoding" or "encoded", with respect to a specified nucleic acid, is meant comprising the information for translation into the specified protein. A nucleic acid encoding a protein may comprise non-translated sequences (e.g., introns) within translated regions of the nucleic acid, or may lack such intervening non-translated sequences (e.g., as in cDNA). The information by which a protein is encoded is specified by the use of codons. Typically, the amino acid sequence is encoded by the nucleic acid using the "universal" genetic code. However, variants of the universal code, such as is present in some plant, animal, and fungal mitochondria, the bacterium Mycoplasma capricolum (Proc. Natl. Acad. Sci. (USA), 82: 2306 2309 (1985)), or the ciliate Macronucleus, may be used when the nucleic acid is expressed using these organisms.

When the nucleic acid is prepared or altered synthetically, advantage can be taken of known codon preferences of the intended host where the nucleic acid is to be expressed.

As used herein, "heterologous" in reference to a nucleic acid is a nucleic acid that originates from a foreign species, or, if from the same species, is substantially modified from its native form in composition and/or genomic locus by deliberate human intervention. For example, a promoter operably linked to a heterologous structural gene is from a species different from that from which the structural gene was derived, or, if from the same species, one or both are substantially modified from their original form. A heterologous protein may originate from a foreign species or, if from the same species, is substantially modified from its original form by deliberate human intervention.

By "host cell" is meant a cell, which contains a vector and supports the replication and/or expression of the expression vector. Host cells may be prokaryotic cells such as E. coli, or eukaryotic cells such as yeast, insect, plant, amphibian, or mammalian cells. Preferably, host cells are bacterial cells to provide for production of the enzyme in large quantities. A particularly preferred bacterial host cell is an E. coli host cell.

The term "hybridization complex" includes reference to a duplex nucleic acid structure formed by two single-stranded nucleic acid sequences selectively hybridized with each other.

The term "introduced" in the context of inserting a nucleic acid into a cell, means "transfection" or "transformation" or "transduction" and includes reference to the incorporation of a nucleic acid into a eukaryotic or prokaryotic cell where the nucleic acid may be incorporated into the genome of the cell (e.g., chromosome, plasmid, plastid or mitochondrial DNA), converted into an autonomous replicon, or transiently expressed (e.g., transfected mRNA).

The terms "isolated" refers to material, such as a nucleic acid or a protein, which is substantially or essentially free from components which normally accompany or interact with it as found in its naturally occurring environment. The isolated material optionally comprises material not found with the material in its natural environment. Nucleic acids, which are "isolated", as defined herein, are also referred to as "heterologous" nucleic acids.

Unless otherwise stated, the term "CAR nucleic acid" means a nucleic acid, including all conservatively modified variants, encoding an CAR polypeptide. The term CAR, unless otherwise stated encompasses CAR and its functional, conservatively modified variants.

As used herein, "nucleic acid" includes reference to a deoxyribonucleotide or ribonucleotide polymer in either single- or double-stranded form, and unless otherwise limited, encompasses known analogues having the essential nature of natural nucleotides in that they hybridize to single-stranded nucleic acids in a manner similar to naturally occurring nucleotides (e.g., peptide nucleic acids).

By "nucleic acid library" is meant a collection of isolated DNA or RNA molecules, which comprise and substantially represent the entire transcribed fraction of a genome of a specified organism. Construction of exemplary nucleic acid libraries, such as genomic and cDNA libraries, is taught in standard molecular biology references such as Berger and Kimmel, Guide to Molecular Cloning Techniques, Methods in Enzymology, Vol. 152, Academic Press, Inc., San Diego, Calif. (Berger); Sambrook et al., Molecular Cloning--A Laboratory Manual, 2nd ed., Vol. 1 3 (1989); and Current Protocols in Molecular Biology, F. M. Ausubel et al., Eds., Current Protocols, a joint venture between Greene Publishing Associates, Inc. and John Wiley & Sons, Inc. (1994 Supplement).

As used herein "operably linked" includes reference to a functional linkage between a first sequence, such as a promoter and a second sequence, wherein the promoter sequence initiates and mediates transcription of the DNA sequence corresponding to the second sequence. Generally, operably linked means that the nucleic acid sequences being linked are contiguous and, where necessary to join two protein coding regions, contiguous and in the same reading frame.

As used herein, "polynucleotide" includes reference to a deoxyribopolynucleotide, ribopolynucleotide, or analogs thereof that have the essential nature of a natural ribonucleotide in that they hybridize, under stringent hybridization conditions, to substantially the same nucleotide sequence as naturally occurring nucleotides and/or allow translation into the same amino acid(s) as the naturally occurring nucleotide(s). A polynucleotide can be full-length or a subsequence of a native or heterologous structural or regulatory gene. Unless otherwise indicated, the term includes reference to the specified sequence as well as the complementary sequence thereof. Thus, DNAs or RNAs with backbones modified for stability or for other reasons are "polynucleotides" as that term is intended herein. Moreover, DNAs or RNAs comprising unusual bases, such as inosine, or modified bases, such as tritylated bases, to name just two examples, are polynucleotides as the term is used herein. It will be appreciated that a great variety of modifications have been made to DNA and RNA that serve many useful purposes known to those of skill in the art. The term polynucleotide as it is employed herein embraces such chemically, enzymatically or metabolically modified forms of polynucleotides, as well as the chemical forms of DNA and RNA characteristic of viruses and cells, including inter alia, simple and complex cells.

The terms "polypeptide", "peptide" and "protein" are used interchangeably herein to refer to a polymer of amino acid residues. The terms apply to amino acid polymers in which one or more amino acid residue is an artificial chemical analogue of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers.

As used herein "promoter" includes reference to a region of DNA upstream from the start of transcription and involved in recognition and binding of RNA polymerase and other proteins to initiate transcription. An "inducible" or "regulatable" promoter is a promoter, which is under environmental control. Examples of environmental conditions that may effect transcription by inducible promoters include anaerobic conditions or the presence of light. Another type of promoter is a developmentally regulated or tissue specific promoter. Tissue preferred, cell type specific, developmentally regulated, and inducible promoters constitute the class of "non-constitutive" promoters. A "constitutive" promoter is a promoter, which is active under most environmental conditions.

The term "CAR polypeptide" refers to one or more amino acid sequences. The term is also inclusive of conservatively modified variants, fragments, homologs, alleles or precursors (e.g., preproproteins or proproteins) thereof. A "CAR protein" comprises a CAR polypeptide.

As used herein "recombinant" includes reference to a cell or vector, that has been modified by the introduction of a heterologous nucleic acid or that the cell is derived from a cell so modified. Thus, for example, recombinant cells express genes that are not found in identical form within the native (non-recombinant) form of the cell or express native genes that are otherwise abnormally expressed, under expressed or not expressed at all as a result of deliberate human intervention. The term "recombinant" as used herein does not encompass the alteration of the cell or vector by naturally occurring events (e.g., spontaneous mutation, natural transformation/transduction/transposition) such as those occurring without deliberate human intervention.

As used herein, a "recombinant expression cassette" is a nucleic acid construct, generated recombinantly or synthetically, with a series of specified nucleic acid elements, which permit transcription of a particular nucleic acid in a target cell. The recombinant expression cassette can be incorporated into a plasmid, chromosome, mitochondrial DNA, plastid DNA, virus, or nucleic acid fragment. Typically, the recombinant expression cassette portion of an expression vector includes, among other sequences, a nucleic acid to be transcribed, and a promoter.

The term "residue" or "amino acid residue" or "amino acid" are used interchangeably herein to refer to an amino acid that is incorporated into a protein, polypeptide, or peptide (collectively "protein"). The amino acid may be a naturally occurring amino acid and, unless otherwise limited, may encompass known analogs of natural amino acids that can function in a similar manner as naturally occurring amino acids.

The term "selectively hybridizes" includes reference to hybridization, under stringent hybridization conditions, of a nucleic acid sequence to a specified nucleic acid target sequence to a detectably greater degree (e.g., at least 2-fold over background) than its hybridization to non-target nucleic acid sequences and to the substantial exclusion of non-target nucleic acids. Selectively hybridizing sequences typically have about at least 40% sequence identity, preferably 60 90% sequence identity, and most preferably 100% sequence identity (i.e., complementary) with each other.

The terms "stringent conditions" or "stringent hybridization conditions" include reference to conditions under which a probe will hybridize to its target sequence, to a detectably greater degree than other sequences (e.g., at least 2-fold over background). Stringent conditions are sequence-dependent and will be different in different circumstances. By controlling the stringency of the hybridization and/or washing conditions, target sequences can be identified which can be up to 100% complementary to the probe (homologous probing). Alternatively, stringency conditions can be adjusted to allow some mismatching in sequences so that lower degrees of similarity are detected (heterologous probing). Optimally, the probe is approximately 500 nucleotides in length, but can vary greatly in length from less than 500 nucleotides to equal to the entire length of the target sequence.

Typically, stringent conditions will be those in which the salt concentration is less than about 1.5 M Na ion, typically about 0.01 to 1.0 M Na ion concentration (or other salts) at pH 7.0 to 8.3 and the temperature is at least about 30.degree. C. for short probes (e.g., 10 to 50 nucleotides) and at least about 60.degree. C. for long probes (e.g., greater than 50 nucleotides). Stringent conditions may also be achieved with the addition of destabilizing agents such as formamide or Denhardt's. Exemplary low stringency conditions include hybridization with a buffer solution of 30 to 35% formamide, 1 M NaCl, 1% SDS (sodium dodecyl sulphate) at 37.degree. C., and a wash in 1.times. to 2.times.SSC (20.times.SSC=3.0 M NaCl/0.3 M trisodium citrate) at 50 to 55.degree. C. Exemplary moderate stringency conditions include hybridization in 40 to 45% formamide, 1 M NaCl, 1% SDS at 37.degree. C., and a wash in 0.5.times. to 1.times.SSC at 55 to 60.degree. C. Exemplary high stringency conditions include hybridization in 50% formamide, 1 M NaCl, 1% SDS at 37.degree. C., and a wash in 0.1.times.SSC at 60 to 65.degree. C. Specificity is typically the function of post-hybridization washes, the critical factors being the ionic strength and temperature of the final wash solution. For DNA-DNA hybrids, the T.sub.m can be approximated from the equation of Meinkoth and Wahl, Anal. Biochem., 138:267 284 (1984): T.sub.m=81.5.degree. C.+16.6 (log M)+0.41 (% GC)-0.61 (% form)-500/L; where M is the molarity of monovalent cations, % GC is the percentage of guanosine and cytosine nucleotides in the DNA, % form is the percentage of formamide in the hybridization solution, and L is the length of the hybrid in base pairs. The T.sub.m is the temperature (under defined ionic strength and pH) at which 50% of a complementary target sequence hybridizes to a perfectly matched probe. T.sub.m is reduced by about 1.degree. C. for each 1% of mismatching; thus, T.sub.m, hybridization and/or wash conditions can be adjusted to hybridize to sequences of the desired identity. For example, if sequences with .gtoreq.90% identity are sought, the T.sub.m can be decreased 10.degree. C. Generally, stringent conditions are selected to be about 5.degree. C. lower than the thermal melting point (T.sub.m) for the specific sequence and its complement at a defined ionic strength and pH. However, severely stringent conditions can utilize a hybridization and/or wash at 1, 2, 3, or 4.degree. C. lower than the thermal melting point (T.sub.m); moderately stringent conditions can utilize a hybridization and/or wash at 6, 7, 8, 9, or 10.degree. C. lower than the thermal melting point (T.sub.m); low stringency conditions can utilize a hybridization and/or wash at 11, 12, 13, 14, 15, or 20.degree. C. lower than the thermal melting point (T.sub.m). Using the equation, hybridization and wash compositions, and desired T.sub.m, those of ordinary skill will understand that variations in the stringency of hybridization and/or wash solutions are inherently described. If the desired degree of mismatching results in a T.sub.m of less than 45.degree. C. (aqueous solution) or 32.degree. C. (formamide solution) it is preferred to increase the SSC concentration so that a higher temperature can be used. An extensive guide to the hybridization of nucleic acids is found in Tijssen, Laboratory Techniques in Biochemistry and Molecular Biology--Hybridization with Nucleic Acid Probes, Part I, Chapter 2 "Overview of principles of hybridization and the strategy of nucleic acid probe assays", Elsevier, New York (1993); and Current Protocols in Molecular Biology, Chapter 2, Ausubel, et al., Eds., Greene Publishing and Wiley-Interscience, New York (1995). Unless otherwise stated, in the present application high stringency is defined as hybridization in 4.times.SSC, 5.times. Denhardt's (5 g Ficoll, 5 g polyvinylpyrrolidone, 5 g bovine serum albumin in 500 ml of water), 0.1 mg/ml boiled salmon sperm DNA, and 25 mM Na phosphate at 65.degree. C., and a wash in 0.1.times.SSC, 0.1% SDS at 65.degree. C.

"Transgenic" is used herein to include any cell, cell line, or tissue, the genotype of which has been altered by the presence of heterologous nucleic acid including those transgenics initially so altered as well as those created by sexual crosses or asexual propagation from the initial transgenic. The term "transgenic" as used herein does not encompass the alteration of the genome (chromosomal or extra-chromosomal) by conventional breeding methods or by naturally occurring events such as random cross-fertilization, non-recombinant viral infection, non-recombinant bacterial transformation, non-recombinant transposition, or spontaneous mutation.

As used herein, "vector" includes reference to a nucleic acid used in transfection of a host cell and into which can be inserted a polynucleotide. Vectors are often replicons. Expression vectors permit transcription of a nucleic acid inserted therein.

The following terms are used to describe the sequence relationships between two or more nucleic acids or polynucleotides or polypeptides: (a) "reference sequence", (b) "comparison window", (c) "sequence identity", (d) "percentage of sequence identity", and (e) "substantial identity".

(a) As used herein, "reference sequence" is a defined sequence used as a basis for sequence comparison. A reference sequence may be a subset or the entirety of a specified sequence; for example, as a segment of a full-length cDNA or gene sequence, or the complete cDNA or gene sequence.

(b) As used herein, "comparison window" means includes reference to a contiguous and specified segment of a polynucleotide sequence, wherein the polynucleotide sequence may be compared to a reference sequence and wherein the portion of the polynucleotide sequence in the comparison window may comprise additions or deletions (i.e., gaps) compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. Generally, the comparison window is at least 20 contiguous nucleotides in length, and optionally can be 30, 40, 50, 100, or longer. Those of skill in the art understand that to avoid a high similarity to a reference sequence due to inclusion of gaps in the polynucleotide sequence a gap penalty is typically introduced and is subtracted from the number of matches.

Methods of alignment of nucleotide and amino acid sequences for comparison are well known in the art. The local homology algorithm (Best Fit) of Smith and Waterman, Adv. Appl. Math may conduct optimal alignment of sequences for comparison. 2: 482 (1981); by the homology alignment algorithm (GAP) of Needleman and Wunsch, J. Mol. Biol. 48: 443 (1970); by the search for similarity method (Tfasta and Fasta) of Pearson and Lipman, Proc. Natl. Acad. Sci. 85: 2444 (1988); by computerized implementations of these algorithms, including, but not limited to: CLUSTAL in the PC/Gene program by Intelligenetics, Mountain View, Calif., GAP, BESTFIT, BLAST, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group (GCG), 575 Science Dr., Madison, Wis., USA; the CLUSTAL program is well described by Higgins and Sharp, Gene 73: 237 244 (1988); Higgins and Sharp, CABIOS 5: 151 153 (1989); Corpet, et al., Nucleic Acids Research 16: 10881 90 (1988); Huang, et a., Computer Applications in the Biosciences 8: 155 65 (1992), and Pearson, et al., Methods in Molecular Biology 24: 307 331 (1994). The preferred program to use for optimal global alignment of multiple sequences is PileUp (Feng and Doolittle, Journal of Molecular Evolution, 25:351 360 (1987) which is similar to the method described by Higgins and Sharp, CABIOS, 5:151 153 (1989) and hereby incorporated by reference). The BLAST family of programs which can be used for database similarity searches includes: BLASTN for nucleotide query sequences against nucleotide database sequences; BLASTX for nucleotide query sequences against protein database sequences; BLASTP for protein query sequences against protein database sequences; TBLASTN for protein query sequences against nucleotide database sequences; and TBLASTX for nucleotide query sequences against nucleotide database sequences. See, Current Protocols in Molecular Biology, Chapter 19, Ausubel, et al., Eds., Greene Publishing and Wiley-Interscience, New York (1995).

GAP uses the algorithm of Needleman and Wunsch (J. Mol. Biol. 48: 443 453, 1970) to find the alignment of two complete sequences that maximizes the number of matches and minimizes the number of gaps. GAP considers all possible alignments and gap positions and creates the alignment with the largest number of matched bases and the fewest gaps. It allows for the provision of a gap creation penalty and a gap extension penalty in units of matched bases. GAP must make a profit of gap creation penalty number of matches for each gap it inserts. If a gap extension penalty greater than zero is chosen, GAP must, in addition, make a profit for each gap inserted of the length of the gap times the gap extension penalty. Default gap creation penalty values and gap extension penalty values in Version 10 of the Wisconsin Genetics Software Package are 8 and 2, respectively. The gap creation and gap extension penalties can be expressed as an integer selected from the group of integers consisting of from 0 to 100. Thus, for example, the gap creation and gap extension penalties can be 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, or greater.

GAP presents one member of the family of best alignments. There may be many members of this family, but no other member has a better quality. GAP displays four figures of merit for alignments: Quality, Ratio, Identity, and Similarity. The Quality is the metric maximized in order to align the sequences. Ratio is the quality divided by the number of bases in the shorter segment. Percent Identity is the percent of the symbols that actually match. Percent Similarity is the percent of the symbols that are similar. Symbols that are across from gaps are ignored. A similarity is scored when the scoring matrix value for a pair of symbols is greater than or equal to 0.50, the similarity threshold. The scoring matrix used in Version 10 of the Wisconsin Genetics Software Package is BLOSUM62 (see Henikoff & Henikoff (1989) Proc. Natl. Acad. Sci. USA 89:10915).

Unless otherwise stated, sequence identity/similarity values provided herein refer to the value obtained using the BLAST 2.0 suite of programs using default parameters. Altschul et al., Nucleic Acids Res. 25:3389 3402 (1997).

As those of ordinary skill in the art will understand, BLAST searches assume that proteins can be modeled as random sequences. However, many real proteins comprise regions of nonrandom sequences, which may be homopolymeric tracts, short-period repeats, or regions enriched in one or more amino acids. Such low-complexity regions may be aligned between unrelated proteins even though other regions of the protein are entirely dissimilar. A number of low-complexity filter programs can be employed to reduce such low-complexity alignments. For example, the SEG (Wooten and Federhen, Comput. Chem., 17:149 163 (1993)) and XNU (Claverie and States, Comput. Chem., 17:191 201 (1993)) low-complexity filters can be employed alone or in combination.

(c) As used herein, "sequence identity" or "identity" in the context of two nucleic acid or polypeptide sequences includes reference to the residues in the two sequences, which are the same when aligned for maximum correspondence over a specified comparison window. When percentage of sequence identity is used in reference to proteins it is recognized that residue positions which are not identical often differ by conservative amino acid substitutions, where amino acid residues are substituted for other amino acid residues with similar chemical properties (e.g. charge or hydrophobicity) and therefore do not change the functional properties of the molecule. Where sequences differ in conservative substitutions, the percent sequence identity may be adjusted upwards to correct for the conservative nature of the substitution. Sequences, which differ by such conservative substitutions, are said to have "sequence similarity" or "similarity". Means for making this adjustment are well known to those of skill in the art. Typically this involves scoring a conservative substitution as a partial rather than a full mismatch, thereby increasing the percentage sequence identity. Thus, for example, where an identical amino acid is given a score of 1 and a non-conservative substitution is given a score of zero, a conservative substitution is given a score between zero and 1. The scoring of conservative substitutions is calculated, e.g., according to the algorithm of Meyers and Miller, Computer Applic. Biol. Sci., 4: 11 17 (1988) e.g., as implemented in the program PC/GENE (Intelligenetics, Mountain View, Calif., USA).

(d) As used herein, "percentage of sequence identity" means the value determined by comparing two optimally aligned sequences over a comparison window, wherein the portion of the polynucleotide sequence in the comparison window may comprise additions or deletions (i.e., gaps) as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the result by 100 to yield the percentage of sequence identity.

(e) (i) The term "substantial identity" of polynucleotide sequences means that a polynucleotide comprises a sequence that has between 50 100% sequence identity, preferably at least 50% sequence identity, preferably at least 60% sequence identity, preferably at least 87%, more preferably at least 90%, more preferably at least 95%, compared to a reference sequence using one of the alignment programs described using standard parameters. One of skill will recognize that these values can be appropriately adjusted to determine corresponding identity of proteins encoded by two nucleotide sequences by taking into account codon degeneracy, amino acid similarity, reading frame positioning and the like. Substantial identity of amino acid sequences for these purposes normally means sequence identity of between 55 100%, preferably at least 75%, preferably at least 80%, more preferably at least 90%, and most preferably at least 95%.

Another indication that nucleotide sequences are substantially identical is if two molecules hybridize to each other under stringent conditions. The degeneracy of the genetic code allows for many amino acids substitutions that lead to variety in the nucleotide sequence that code for the same amino acid, hence it is possible that the DNA sequence could code for the same polypeptide but not hybridize to each other under stringent conditions. This may occur, e.g., when a copy of a nucleic acid is created using the maximum codon degeneracy permitted by the genetic code. One indication that two nucleic acid sequences are substantially identical is that the polypeptide, which the first nucleic acid encodes, is immunologically cross reactive with the polypeptide encoded by the second nucleic acid.

(e) (ii) The terms "substantial identity" in the context of a peptide indicates that a peptide comprises a sequence with between 55 100% sequence identity to a reference sequence preferably at least 55% sequence identity, preferably 60% preferably 70%, more preferably 80%, most preferably at least 90% or 95% sequence identity to the reference sequence over a specified comparison window. Preferably, optimal alignment is conducted using the homology alignment algorithm of Needleman and Wunsch, J. Mol. Biol. 48:443 (1970). An indication that two peptide sequences are substantially identical is that one peptide is immunologically reactive with antibodies raised against the second peptide. Thus, a peptide is substantially identical to a second peptide, for example, where the two peptides differ only by a conservative substitution. In addition, a peptide can be substantially identical to a second peptide when they differ by a non-conservative change if the epitope that the antibody recognizes is substantially identical. Peptides, which are "substantially similar" share sequences as, noted above except that residue positions, which are not identical, may differ by conservative amino acid changes.

Carboxylic acid reductase (CAR) catalyzes the first and rate limiting step in the reduction of carboxylic acids to aldehydes, and later alcohols. According to the invention, analysis of a cloned 6.9 Kb sequence revealed that the entire open reading frame of Nocardia CAR and its 5' and 3' flanking regions had been cloned. ATG was identified as the translation start codon by matching the N-terminal amino acid sequence from purified Nocardia CAR (23) with an amino acid sequence deduced from the DNA sequence. The assignment of ATG as the start codon is supported by 5' flank region analysis: 6 bp upstream from the start codon ATG lies a conserved Streptomyces ribosomal binding site (GGGAGG) (27, 35). The 2.5 Kb sequence upstream of CAR showed fair homology to a putative transmembrane efflux protein (33% identity) in S. avermitilis, and a putative efflux protein (32% identity) in M. tuberculosis. The sequence downstream of Nocardia car showed 40%, 35%, 34% and 28% identities to putative membrane proteins in Corynebacterium efficiens, M. tuberculosis, M. leprae, and S. coelicolor, respectively. Although the CAR gene was flanked by genes encoding membrane proteins, the actual function of CAR in Nocardia remains unknown at this time.

BLAST analysis also showed that CAR contained two major domains and a possible phosphopantetheine attachment site. The N-terminal domain (aa 90 544) showed high homology to AMP-binding proteins. The C-terminal showed high homology to NADPH binding proteins. If a 4'-phosphopantetheine prosthetic group exists in active CAR, it likely acts as a "swinging arm" for transferring acyl-AMP intermediates to the C-terminal reductase domain. This arrangement of the CAR protein would reflect its sequential catalytic mechanism wherein the N-terminal domain catalyzes substrate activation by formation of an initial acyl-AMP intermediate, while the C-terminal portion then catalyzes the reduction of acyl-AMP by cofactor NADPH to finish a catalytic cycle. The existence of a possible 4'-phosphopantetheine prosthetic group for the catalytic process remains to be shown.

By BLAST analysis, the deduced amino acid sequence of Nocardia CAR showed high similarity to those of the putative enzymes in M. tuberculosis (fadD9, 61% identity), M. leprae (acyl-CoA synthetase, 57% identity), M. smegmatis (unnamed hypothetical protein on contig:3313, 61.8% identity), M. bovis strain BCG (unnamed hypothetical protein at bases 2,885,319 2,888.822, 60.3% identity), suggesting that possible functions of these proteins may relate to carboxylic acid reduction.

The present invention provides, inter alia, isolated nucleic acids of RNA, DNA, and analogs and/or chimeras thereof, comprising a CAR nucleic acid.

The present invention also includes polynucleotides optimized for expression in different organisms. For example, for expression of the polynucleotide in a maize plant, the sequence can be altered to account for specific codon preferences and to alter GC content as according to Murray et al, supra. Maize codon usage for 28 genes from maize plants is listed in Table 4 of Murray, et al., supra.

The CAR nucleic acids of the present invention comprise isolated CAR nucleic acid sequences which, are inclusive of:

(a) an isolated polynucleotide encoding a polypeptide of the present invention; (b) a polynucleotide having at least 80%, 90% or 95% identity to a polynucleotide of the present invention; (c) a polynucleotide comprising at least 25 nucleotides in length which hybridizes under high stringency conditions to a polynucleotide of the present invention; (d) a polynucleotide comprising a polynucleotide of the present invention; and (e) a polynucleotide which is complementary to the polynucleotide of (a) to (d).

The following description sets forth the general procedures involved in practicing the present invention. To the extent that specific materials are mentioned, it is merely for purposes of illustration and is not intended to limit the invention. Unless otherwise specified, general cloning procedures, such as those set forth in Sambrook et al., Molecular Cloning, Cold Spring Harbor Laboratory (1989) (hereinafter "Sambrook et al.") or Ausubel et al. (eds) Current Protocols in Molecular Biology, John Wiley & Sons (1999) (hereinafter "Ausubel et al." are used.

A. Preparation of CAR, Antibodies Specific for CAR and Nucleic Acid Molecules Encoding CAR

1. Proteins and Antibodies

CAR may be prepared in a variety of ways, according to a variety of methods that have been developed for purifying CAR from bacteria which are detailed in the materials incorporated herein by reference. Alternatively, the availability of amino acid sequence information, such as (SEQ ID NO: 2), enables the isolation of nucleic acid molecules encoding CAR. This may be accomplished using anti-CAR antibodies to screen a cDNA expression library from a selected species, according to methods well known in the art. Alternatively, a series of degenerate oligonucleotide probes encoding parts or all of (SEQ ID NO: 1) FIG. 2 may be used to screen cDNA or genomic libraries, as described in greater detail below.

Once obtained, a cDNA or gene may be cloned into an appropriate in vitro transcription vector, such a pSP64 or pSP65 for in vitro transcription, followed by cell-free translation in a suitable cell-free translation system, such as wheat germ or rabbit reticulocytes. In vitro transcription and translation systems are commercially available, e.g., from Promega Biotech, Madison, Wis. or BRL, Rockville, Md. The pCITE in vitro translation system (Novagen) also may be utilized.

According to a preferred embodiment, larger quantities of the proteins may be produced by expression in a suitable procaryotic or eucaryotic system. This is particularly beneficial for CAR as Nocardia sp. are difficult to propagate and maintain in culture. For example, part or all of a CAR-encoding DNA molecule may be inserted into a vector adapted for expression in a bacterial cell (such as E. coli) or a yeast cell (such as Saccharomyces cerevisiae), or a mammalian cell. Such vectors comprise the regulatory elements necessary for expression of the DNA in the host cell, positioned in such a manner as to permit expression of the DNA in the host cell. Such regulatory elements required for expression include operably linked promoter sequences, transcription initiation sequences and, optionally, enhancer sequences.

CAR produced by gene expression in a recombinant procaryotic or eukaryotic system may be purified according to methods known in the art and incorporated herein. In a preferred embodiment, a commercially available expression/secretion system can be used, whereby the recombinant protein is expressed and thereafter secreted from the host cell, to be easily purified from the surrounding medium. If expression/secretion vectors are not used, an alternative approach involves purifying the recombinant protein by affinity separation, such as by immunological interaction with antibodies that bind specifically to the recombinant protein or with expression/secretion systems (e.g. a C-terminal tag on a secreted protein). Such methods are commonly used by skilled practitioners.

The present invention also provides antibodies capable of binding to CAR from one or more selected species. Polyclonal or monoclonal antibodies directed toward part or all of a selected CAR may be prepared according to standard methods. Monoclonal antibodies may be prepared according to general methods of Kohler and Milstein, following standard protocols. In a preferred embodiment, antibodies are prepared, which react immunospecifically with selected epitopes of CAR distinguishing it from other enzymes.

2. Nucleic Acid Molecules

Once sequence information is obtained, nucleic acid molecules encoding CAR may be prepared by two general methods: (1) they may be synthesized from appropriate nucleotide triphosphates, or (2) they may be isolated from biological sources. Both methods utilize protocols well known in the art.

The availability of nucleotide sequence information enables preparation of an isolated nucleic acid molecule of the invention by oligonucleotide synthesis. Synthetic oligonucleotides may be prepared by the phosphoramadite method employed in the Applied Biosystems 38A DNA Synthesizer or similar devices. The resultant construct may be purified according to methods known in the art, such as high performance liquid chromatography (HPLC). Long, double-stranded polynucleotides, such as a DNA molecule of the present invention, must be synthesized in stages, due to the size limitations inherent in current oligonucleotide synthetic methods. Thus, for example, a long double-stranded molecule may be synthesized as several smaller segments of appropriate complementarity. Complementary segments thus produced may be annealed such that each segment possesses appropriate cohesive termini for attachment of an adjacent segment. Adjacent segments may be ligated by annealing cohesive termini in the presence of DNA ligase to construct an entire long double-stranded molecule. A synthetic DNA molecule so constructed may then be cloned and amplified in an appropriate vector.

Nucleic acid molecules encoding CAR also may be isolated from microorganisms of interest using methods well known in the art. Nucleic acid molecules from a selected species may be isolated by screening cDNA or genomic libraries with oligonucleotides designed to match a nucleic acid sequence specific to a CAR-encoding gene. If the gene from a species is desired, the genomic library is screened. Alternatively, if the protein coding sequence is of particular interest, the cDNA library is screened. In positions of degeneracy, where more than one nucleic acid residue could be used to encode the appropriate amino acid residue, all the appropriate nucleic acids residues may be incorporated to create a mixed oligonucleotide population, or a neutral base such as inosine may be used. The strategy of oligonucleotide design is well known in the art (see also Sambrook et al., Molecular Cloning, 1989, Cold Spring Harbor Press, Cold Spring Harbor N.Y.).

Alternatively, PCR (polymerase chain reaction) primers may be designed by the above method to encode a portion of CAR protein, and these primers used to amplify nucleic acids from isolated cDNA or genomic DNA. In a preferred embodiment, the oligonucleotides used to isolate CAR-encoding nucleic acids ar