Lipoxygenase proteins and nucleic acids

Abstract

Isolated and purified lipoxygenase proteins and nucleic acids are described. Particularly, a novel human 15(S) lipoxygenase (15-Lox-2) protein and cDNA and a cDNA for mouse 8S-lipoxygenase are described. Recombinant host cells, recombinant nucleic acids and recombinant proteins are also described, along with methods of producing each. Isolated and purified antibodies to 15-Lox-2 and 8-Lox, and methods of producing the same, are also described.

What is claimed is:  
     
         1 . A nucleic acid segment comprising an isolated gene encoding a lipoxygenase, said lipoxygenase containing an iron ligand comprising a serine.  
     
     
         2 . The nucleic acid segment of  claim 1 , wherein said isolated gene encodes a polypeptide having an in vivo molecular weight of about 76 KD when measured by SDS-PAGE.  
     
     
         3 . The nucleic acid segment of  claim 1 , wherein the encoded lipoxygenase converts arachidonic acid exclusively to 15S-hydroperoxyeicosatetraenoic acid or Converts arachidonic acid exclusively to 8S-hydroperoxyeicosatetraenoic acid.  
     
     
         4 . The nucleic acid segment of  claim 1 , wherein the isolated gene encodes 15-Lox-2 or 8-Lox.  
     
     
         5 . The nucleic acid segment of  claim 1 , further defined as a DNA segment.  
     
     
         6 . A recombinant host cell comprising the nucleic acid segment of  claim 1 .  
     
     
         7 . The nucleic acid segment of  claim 4 , wherein the isolated gene encodes 15-Lox-2.  
     
     
         8 . The nucleic acid segment of  claim 4 , wherein the isolated gene encodes 8-Lox.  
     
     
         9 . The nucleic acid segment of  claim 7 , wherein the isolated gene encodes 15-Lox-2 comprising the amino acid sequence of SEQ ID NO:2.  
     
     
         10 . The nucleic acid segment of  claim 9 , further defined as comprising the 15-Lox-2-coding nucleic acid sequence of SEQ ID NO:1.  
     
     
         11 . The nucleic acid segment of  claim 8 , wherein the isolated gene encodes 8-Lox comprising the amino acid sequence of SEQ ID NO:4.  
     
     
         12 . The nucleic acid segment of  claim 11 , further defined as comprising 8-Lox-coding nucleic acid sequence of SEQ ID NO:3.  
     
     
         13 . The nucleic acid segment of  claim 5 , wherein the isolated gene is positioned under the control of a promoter.  
     
     
         14 . The nucleic acid segment of  claim 13 , further defined as a recombinant vector which comprises the isolated gene.  
     
     
         15 . The nucleic acid segment of  claim 14 , wherein the vector is a recombinant expression vector.  
     
     
         16 . The recombinant host cell of  claim 6 , wherein the host cell is a procaryotic cell.  
     
     
         17 . The recombinant host cell of  claim 6 , wherein the host cell is a eucaryotic cell.  
     
     
         18 . A nucleic acid segment which comprises at least a 10 nucleotide long contiguous stretch of the nucleic acid sequence of SEQ ID NO:1 or SEQ ID NO:3.  
     
     
         19 . The nucleic acid segment of  claim 18 , further defined as comprising at least a 15 nucleotide long contiguous stretch of the nucleic acid sequence of SEQ ID NO:1 or SEQ ID NO:3.  
     
     
         20 . The nucleic acid segment of  claim 19 , further defined as comprising at least a 20 nucleotide long contiguous stretch of the nucleic acid sequence of SEQ ID NO:1 or SEQ ID NO:3.  
     
     
         21 . The nucleic acid segment of  claim 19 , further defined as a nucleic acid fragment of up to 10,000 basepairs in length.  
     
     
         22 . The nucleic acid segment of  claim 20 , further defined as comprising at least a 30 nucleotide long contiguous stretch of the nucleic acid sequence of SEQ ID NO:1 or SEQ ID NO:3.  
     
     
         23 . The nucleic acid segment of  claim 22 , further defined as comprising at least a 50 nucleotide long contiguous stretch of the nucleic acid sequence of SEQ ID NO:1 or SEQ ID NO:3.  
     
     
         24 . The nucleic acid segment of  claim 23 , further defined as comprising at least a 100 nucleotide long contiguous stretch of the nucleic acid sequence of SEQ ID NO:1 or SEQ ID NO:3.  
     
     
         25 . The nucleic acid segment of  claim 24 , further defined as comprising at least a 1000 nucleotide long contiguous stretch of the nucleic acid sequence of SEQ ID NO:1 or SEQ ID NO:3.  
     
     
         26 . The nucleic acid segment of  claim 25 , further defined as having the nucleic acid sequence of SEQ ID NO:1 or SEQ ID NO:3.  
     
     
         27 . The nucleic acid segment of  claim 21 , further defined as a nucleic acid fragment of up to 1,000 basepairs in length.  
     
     
         28 . The nucleic acid segment of  claim 27 , further defined as a nucleic acid fragment of up to 500 basepairs in length.  
     
     
         29 . The nucleic acid segment of  claim 28 , further defined as a nucleic acid fragment of up to 50 basepairs in length.  
     
     
         30 . A method of preparing a lipoxygenase polypeptide, comprising: transforming a cell with the nucleic acid of  claim 1  to produce a lipoxygenase under conditions suitable for the expression of said polypeptide.  
     
     
         31 . A process of detecting in a sample an RNA that encodes the lipoxygenase polypeptide encoded by the nucleic acid of  claim 1 , said process comprising the steps of: 
 (a) contacting said sample under hybridizing conditions with the nucleic acid segment of  claim 1  to form a duplex; and    (b) detecting the presence of said duplex.    
     
     
         32 . An isolated and purified biologically active lipoxygenase polypeptide capable of converting arachidonic acid exclusively to 15S-hydroperoxyeicosatetraenoic acid, said lipoxygenase containing an iron ligand comprising a serine.  
     
     
         33 . A polypeptide of  claim 32 , wherein said polypeptide has an in vivo molecular weight of about 76 KD when measured by SDS-PAGE.  
     
     
         34 . A polypeptide of  claim 32 , further comprising an amino acid sequence W-L-L-A-K (SEQ ID NO:5) and an amino acid sequence G-Q-Y-D-W (SEQ ID NO:35), the amino acid sequence W-L-L-A-K (SEQ ID NO:5) positioned upstream from the amino acid sequence G-Q-Y-D-W (SEQ ID NO:35) along the polypeptide.  
     
     
         35 . A polypeptide according to  claim 32 , wherein the polypeptide comprises a 15-Lox-2.  
     
     
         36 . A polypeptide according to  claim 35 , wherein the 15-Lox-2 comprises the amino acid sequence of SEQ ID NO:2.  
     
     
         37 . A polypeptide according to  claim 32 , modified to be in detectably labeled form.  
     
     
         38 . An isolated and purified antibody capable of specifically binding to the polypeptide of  claim 32 .  
     
     
         39 . The antibody of  claim 38  which is a monoclonal antibody.  
     
     
         40 . The antibody of  claim 38  which is a polyclonal antibody.  
     
     
         41 . A hybridoma cell line which produces the monoclonal antibody of  claim 39 .  
     
     
         42 . An isolated and purified antibody capable of neutralizing the biological activity of the polypeptide of  claim 32 .  
     
     
         43 . The antibody of  claim 42  which is a monoclonal antibody.  
     
     
         44 . The antibody of  claim 42  which is a polyclonal antibody.  
     
     
         45 . A hybridoma cell line which produces the monoclonal antibody of  claim 43 .  
     
     
         46 . A process of producing an antibody immunoreactive with a lipoxygenase polypeptide, the process comprising steps of 
 (a) transfecting a recombinant host cell with the a polynucleotide of  claim 1 , which encodes a lipoxygenase polypeptide;    (b) culturing the host cell under conditions sufficient for expression of the polypeptide;    (c) recovering the polypeptide; and    (d) preparing the antibody to the polypeptide.    
     
     
         47 . The process of  claim 46 , wherein the polypeptide comprises SEQ ID NO:2.  
     
     
         48 . The process of  claim 46 , wherein the poynucleotide comprises SEQ ID NO:1 or comprises SEQ ID NO:3.  
     
     
         49 . An antibody produced by the process of  claim 46 .  
     
     
         50 . A process of detecting a lipoxygenase poypeptide, the process comprising immunoreacting the polypeptide with an antibody prepared according the process of  claim 46  to form an antibody-polypeptide conjugate, and detecting the conjugate.  
     
     
         51 . A process of detecting a messenger RNA transcript that encodes a lipoxygenase polypeptide, the process comprising the steps of hybridizing the messenger RNA transcript with the polynucleotide of  claim 1  to form a duplex; and detecting the duplex.  
     
     
         52 . A process of detecting a DNA molecule that encodes a lipoxygenase polypeptide, the process comprising the steps of hybridizing DNA molecules with the polynucleotide of  claim 1  to form a duplex; and detecting the duplex.  
     
     
         53 . A diagnostic assay kit for detecting the presence of a lipoxygenase polypeptide in a biological sample, the kit comprising a first container containing a first antibody capable of immunoreacting with a lipoxygenase polypeptide encoded by the polynucleotide of  claim 1 , wherein the first antibody is present in an amount sufficient to perform at least one assay.  
     
     
         54 . An assay kit of  claim 53 , further comprising a second container containing a second antibody that immunoreacts with the first antibody.  
     
     
         55 . An assay kit of  claim 54 , wherein the first antibody and the second antibody comprise monoclonal antibodies.  
     
     
         56 . An assay kit of  claim 55 , wherein the first antibody is affixed to a solid support.  
     
     
         57 . An assay kit of  claim 55 , wherein the first and second antibodies each comprise an indicator.  
     
     
         58 . An assay kit of  claim 57 , wherein the indicator is a radioactive label or an enzyme.  
     
     
         59 . A diagnostic assay kit for detecting the presence, in biological samples, of a lipoxygenase polypeptide, the kit comprising a first container that contains a polynucleotide identical or complimentary to a segment of at least ten contiguous nucleotide bases of the polynucleotide of  claim 1 .  
     
     
         60 . A diagnostic assay kit for detecting the presence, in a biological sample, of an antibody immunoreactive with a lipoxygenase polypeptide, the kit comprising a first container containing a lipoxygenase polypeptide encoded by the polynucleotide of  claim 1  that immunoreacts with the antibody, with the polypeptide present in an amount sufficient to perform at least one assay.  
     
     
         61 . A screening assay for identifying a compound that affects arachidonic acid metabolism in a cell, comprising the steps of: 
 (a) establishing replicate test and control cultures of cells that express a lipoxygenase polypeptide encoded by the polynucleotide of  claim 1;     (b) administering a candidate compound to the cells in the test culture but not the control culture;    (c) measuring hydroperoxyeicosatetraenoic acid levels in the test and the control cultures; and    (d) determining that the candidate compound affects arachidonic acid metabolism in a cell if the hydroperoxyeicosatetraenoic acid level measured for the test culture is less or greater than the hydroperoxyeicosatetraenoic acid level measured for the control culture.    
     
     
         62 . An assay of  claim 61 , wherein the lipoxygenase polypeptide comprises 15-Lox-2.  
     
     
         63 . An assay of  claim 61 , wherein the lipoxygenase polypeptide comprises 8-Lox.