Lactobacillus acidophilus nucleic acid sequences encoding protease homologues and uses therefore

Abstract

Protease-like nucleic acid molecules and polypeptides and fragments and variants thereof are disclosed in the current invention. In addition, protease-like fusion proteins, antigenic peptides, and anti-protease-like antibodies are encompassed. The invention also provides vectors containing a nucleic acid molecule of the invention and cells into which the vectors have been introduced. Methods for producing the polypeptides and methods of use for the polypeptides of the invention are further disclosed.

1 . An isolated nucleic acid molecule selected from the group consisting of:
 a) a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO:1, 3, 5, 7, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 36, 38, 40, 42, 44, 46, 49, 51, 53, 57, 59, 61, 63, 65, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133 and/or 146, or a complement thereof;   b) a nucleic acid molecule comprising a nucleotide sequence having at least 90% sequence identity to the nucleotide sequence of SEQ ID NO:1, 3, 5, 7, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 49, 51, 53, 57, 59, 61, 63, 65, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133 and/or 146, or a complement thereof;   c) a nucleic acid molecule that encodes a polypeptide comprising the amino acid sequence of SEQ ID NO:2, 4, 6, 8, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 37, 39, 41, 43, 45, 47, 48, 50, 52, 54, 58, 60, 62, 64, 66, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, and/or 147;   d) a nucleic acid molecule comprising a nucleotide sequence that encodes a polypeptide having at least 90% amino acid sequence identity to the amino acid sequence of SEQ ID NO:2, 4, 6, 8, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 37, 39, 41, 43, 45, 47, 48, 50, 52, 54, 58, 60, 62, 64, 66, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, and/or 147; and,   e) a nucleic acid molecule that hybridizes under stringent conditions to any of a)-d).   
     
     
         2 . A vector comprising the nucleic acid molecule of  claim 1 . 
     
     
         3 . The vector of  claim 2 , further comprising a nucleotide sequence encoding a heterologous polypeptide. 
     
     
         4 . A cell comprising the vector of  claim 2 . 
     
     
         5 . The cell of  claim 4  that is a bacterial cell. 
     
     
         6 . An isolated polypeptide selected from the group consisting of:
 a) a polypeptide comprising the amino acid sequence of SEQ ID NO:2, 4, 6, 8, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 37, 39, 41, 43, 45, 47, 48, 50, 52, 54, 58, 60, 62, 64, 66, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, and/or 147;   b) a polypeptide encoded by the nucleotide sequence of SEQ ID NO:1, 3, 5, 7, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 36, 38, 40, 42, 44, 46, 49, 51, 53, 57, 59, 61, 63, 65, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, and/or 146;   c) a polypeptide comprising an amino acid sequence having at least 90% sequence identity with an amino acid sequence of SEQ ID NO:2, 4, 6, 8, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 37, 39, 41, 43, 45, 47, 48, 50, 52, 54, 58, 60, 62, 64, 66, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, and/or 147; and,   d) a polypeptide encoded by a nucleotide sequence having at least 90% sequence identity to the nucleotide sequence of SEQ ID NO:1, 3, 5, 7, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 36, 38, 40, 42, 44, 46, 49, 51, 53, 57, 59, 61, 63, 65, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, and/or 146.   
     
     
         7 . The polypeptide of  claim 6  further comprising a heterologous amino acid sequence. 
     
     
         8 . An antibody that selectively binds to the polypeptide of  claim 6 . 
     
     
         9 . A method for producing a polypeptide, comprising culturing the cell of  claim 4  under conditions in which a nucleic acid molecule encoding the polypeptide is expressed, said polypeptide being selected from the group consisting of:
 a) a polypeptide comprising the amino acid sequence of SEQ ID NO:2, 4, 6, 8, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 37, 39, 41, 43, 45, 47, 48, 50, 52, 54, 58, 60, 62, 64, 66, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, and/or 147; 
 b) a polypeptide encoded by the nucleotide sequence of SEQ ID NO:1, 3, 5, 7, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 36, 38, 40, 42, 44, 46, 49, 51, 53, 57, 59, 61, 63, 65, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, and/or 146; 
 c) a polypeptide comprising an amino acid sequence having at least 90% sequence identity with an amino acid sequence of SEQ ID NO:2, 4, 6, 8, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 37, 39, 41, 43, 45, 47, 48, 50, 52, 54, 58, 60, 62, 64, 66, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, and/or 147; and, 
 d) a polypeptide encoded by a nucleotide sequence having at least 90% sequence identity to the nucleotide sequence of SEQ ID NO:1, 3, 5, 7, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 36, 38, 40, 42, 44, 46, 49, 51, 53, 57, 59, 61, 63, 65, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, and/or 146. 
 
     
     
         10 . A method for modulating the growth rate of a bacterium, comprising introducing into said bacterium the nucleic acid molecule of  claim 1 . 
     
     
         11 . A method for modulating the acidification rate of a milk product fermented by lactic acid bacteria, comprising introducing into said bacteria the nucleic acid molecule of  claim 1 . 
     
     
         12 . A method for modulating the rate of cheese ripening, comprising fermenting milk used for cheese in the presence of an organism that contains the nucleic acid molecule of  claim 1 . 
     
     
         13 . A method for modulating flavor diversification of fermented milk products, comprising fermenting milk in the presence of an organism that contains the nucleic acid molecule of  claim 1 . 
     
     
         14 . A biologically pure culture of a lactic acid bacterium strain, wherein said strain comprises a nucleotide sequence encoding the amino acid sequence of SEQ ID NO:2, 4, 6, 8, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 37, 39, 41, 43, 45, 47, 48, 50, 52, 54, 58, 60, 62, 64, 66, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, and/or 147. 
     
     
         15 . A food containing the strain of  claim 14 . 
     
     
         16 . The food of  claim 15  wherein the food is selected from the group consisting of milk, yogurt, curd, cheese, fermented milks, ice-creams, fermented cereal based products, milk based powders, infant formulae, tablets, liquid bacterial suspensions, dried oral supplement, and liquid oral supplements.