Thermostable luciferases and methods of production

Abstract

Luciferase enzymes with greatly increased thermostability, e.g., at least half lives of 2 hours at 50° C., cDNAs encoding the novel luciferases, and hosts transformed to express the luciferases, are disclosed. Methods of producing the luciferases include recursive mutagenesis. The luciferases are used in conventional methods, some employing kits.

1 . A thermostable beetle luciferase that retains at least 50% activity in aqueous solution for at least 2 hours at a temperature of about 50° C., wherein the thermostable beetle luciferase has at least one amino acid substitution at an amino acid residue corresponding to residue 2, 27, 91, 144, 183, 220, 262, 353, 356, 386, 399, 412, 413 or 499 of SEQ ID NO:37, or a combination thereof. 
     
     
         2 . The thermostable luciferase of  claim 1  which retains activity for at least 100 days at a temperature of about 22° C. 
     
     
         3 . The thermostable luciferase of  claim 1  which retains activity for at least 12 hours at a temperature of about 37° C. 
     
     
         4 . The thermostable luciferase of  claim 1  which retains activity for at least 2 hours at a temperature of about 65° C. 
     
     
         5 . The thermostable luciferase of  claim 1  which comprises a plurality of amino acid substitutions. 
     
     
         6 . The thermostable luciferase of  claim 1  which retains activity for at least 5 hours at a temperature of about 50° C. 
     
     
         7 . The thermostable luciferase of  claim 1  which retains activity for at least 10 hours at a temperature of about 50° C. 
     
     
         8 . The thermostable luciferase of  claim 1  which retains activity for at least 2 hours at a temperature of about 60° C. 
     
     
         9 . The thermostable luciferase of  claim 1  which retains activity for at least 5 hours at a temperature of about 60° C. 
     
     
         10 . The thermostable luciferase of  claim 1  which retains activity for at least 24 hours at a temperature of about 37° C. 
     
     
         11 . The thermostable luciferase of  claim 1  which retains activity for at least 2 days at a temperature of about 37° C. 
     
     
         12 . The thermostable luciferase of  claim 1  which retains activity for at least 200 days at a temperature of about 22° C. 
     
     
         13 . The thermostable luciferase of  claim 1  which retains activity for at least 500 days at a temperature of about 22° C. 
     
     
         14 . The thermostable luciferase of  claim 1  which retains activity for at least 800 days at a temperature of about 22° C. 
     
     
         15 . The thermostable luciferase of  claim 1  which retains activity for at least 6 hours at a temperature of about 65° C. 
     
     
         16 . The thermostable luciferase of  claim 1  which retains activity for at least 2 days at a temperature of about 65° C. 
     
     
         17 . The thermostable luciferase of  claim 1  which retains at least 90% activity for at least 40 days at a temperature of about 22° C. 
     
     
         18 . The thermostable luciferase of  claim 1  wherein the substitution is at a consensus site. 
     
     
         19 . The thermostable luciferase of  claim 1  wherein the substitution is to a consensus amino acid. 
     
     
         20 . The thermostable luciferase of  claim 1  which has at least one of the amino acid substitutions of SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:44, SEQ ID NO:47, or SEQ ID NO:45. 
     
     
         21 . The thermostable luciferase of  claim 1  which has at least one of the amino acid substitutions of SEQ ID NO:14, SEQ ID NO:19, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:44, SEQ ID NO:47, or SEQ ID NO:45. 
     
     
         22 . The thermostable luciferase of  claim 1  which further comprises increased luminescence intensity or signal stability relative to the reference beetle luciferase. 
     
     
         23 . The thermostable luciferase of  claim 1  which further comprises a decreased K m . 
     
     
         24 . An isolated and purified nucleic acid molecule comprising a nucleic acid segment encoding the thermostable luciferase of  claim 1 . 
     
     
         25 . The isolated and purified nucleic acid molecule of  claim 24  comprising a nucleic acid segment comprising SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:42, SEQ ID NO:46, or SEQ ID NO:43. 
     
     
         26 . The isolated and purified nucleic acid molecule of  claim 24  comprising a nucleic acid segment comprising SEQ ID NO:1, SEQ ID NO:6, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:42, SEQ ID NO:46, or SEQ ID NO:43. 
     
     
         27 . A vector containing the nucleic acid molecule of  claim 24 . 
     
     
         28 . A host cell, the genome of which is augmented with the nucleic acid molecule of  claim 24 . 
     
     
         29 . A solid substrate comprising the luciferase of  claim 1 . 
     
     
         30 . A fusion protein comprising the luciferase of  claim 1 . 
     
     
         31 . An isolated and purified nucleic acid molecule comprising a nucleic acid segment encoding the fusion protein of  claim 30 . 
     
     
         32 . A method of using a luciferase, comprising: linking an agent with the luciferase of  claim 1  so as to yield a labeled agent. 
     
     
         33 . A method of using a vector encoding a luciferase, comprising:
 a) introducing the vector of  claim 27  into a host cell; and   b) detecting or determining the presence of luciferase in the host cell.   
     
     
         34 . A kit comprising: a container comprising the luciferase of  claim 1 . 
     
     
         35 . The kit of  claim 34  wherein the container comprises an aqueous mixture comprising the luciferase. 
     
     
         36 . The kit of  claim 34  wherein the container comprises lyophilized luciferase. 
     
     
         37 . The kit of  claim 34  further comprising instruction means directing the user to correlate the luciferase activity in the sample with the level of ATP in the sample. 
     
     
         38 . The kit of  claim 34  or  37  further comprising a container comprising luciferin. 
     
     
         39 . The kit of  claim 34  or  37  wherein the container further comprises luciferin. 
     
     
         40 . The kit of  claim 38  wherein the container comprising luciferin comprises lyophilized luciferin. 
     
     
         41 . The kit of  claim 39  wherein the luciferin comprises lyophilized luciferin. 
     
     
         42 . The kit of  claim 37  further comprising packaging material enclosing, separately packaged, the container and instruction means directing the user to correlate the luciferase activity in the sample with the level of ATP in the sample. 
     
     
         43 . The kit of  claim 37  further comprising packaging material enclosing, separately packaged, the container and instruction means directing the user to correlate the luciferase activity in the sample with the level or presence of an agent in the sample that produces ATP. 
     
     
         44 . The kit of  claim 34  further comprising instruction means directing the user to correlate the luciferase activity in the sample with the level or presence of an agent in the sample that produces ATP. 
     
     
         45 . The kit of  claim 44  further comprising packaging material enclosing, separately packaged, the container and instruction means directing the user to correlate the luciferase activity in the sample with the level or presence of the molecule. 
     
     
         46 . A method to prepare thermostable luciferases, comprising:
 a) selecting one or more isolated polynucleotide sequences encoding thermostable luciferases which have one or more other desired enzymological properties from a first population of polynucleotide sequences obtained from a first isolated polynucleotide sequence encoding a luciferase subjected to conditions that yield nucleotide mutations, wherein the one or more isolated polynucleotide sequences encode thermostable luciferases which have increased thermostability and one or more enhanced desired enzymological properties relative to the first isolated polynucleotide sequence;   b) mutating the selected isolated polynucleotide sequence to yield a second population of polynucleotide sequences; and   c) repeating step a) and step b) so as to yield a further polynucleotide sequence encoding a thermostable luciferase comprising a plurality of amino acid substitutions and having at least one enhanced enzymological desired property relative to the luciferase encoded by the first polynucleotide sequence.   
     
     
         47 . A method to prepare an enzyme having enhanced enzymological properties, comprising:
 a) selecting one or more isolated polynucleotide sequences encoding an enzyme which has at least one enhanced enzymological property from a first population of polynucleotide sequences obtained from a first isolated polynucleotide sequence encoding an enzyme subjected to conditions that yield nucleotide mutations, wherein the enzyme encoded by the one or more selected isolated polynucleotide sequences has at least one enhanced enzymological property relative to the enzyme encoded by the first isolated polynucleotide sequence;   b) mutating the selected isolated polynucleotide sequence to yield a second population of polynucleotide sequences, wherein the selected isolated polynucleotide sequence is subjected to oligonucleotide mediated mutagenesis with at least one oligonucleotide comprising a codon that encodes a consensus amino acid which is not present in the first polynucleotide sequence; and   c) repeating step a) and step b) so as to yield a further polynucleotide sequence encoding an enzyme having at least one enhanced enzymological property and comprising a plurality of amino acid substitutions relative to the enzyme encoded by the first polynucleotide sequence.   
     
     
         48 . A method to prepare an enzyme that is resistance to an inhibitor, comprising:
 a) selecting one or more isolated polynucleotide sequences encoding an enzyme which is resistance to an inhibitor from a first population of polynucleotide sequences obtained from a first isolated polynucleotide sequence encoding an enzyme subjected to conditions that yield nucleotide mutations, wherein the enzyme encoded by the one or more selected isolated polynucleotide sequences has increased resistance to an inhibitor relative to the enzyme encoded by the first isolated polynucleotide sequence;   b) mutating the selected isolated polynucleotide sequence to yield a second population of polynucleotide sequences; and   c) repeating step a) and step b) so as to yield a further polynucleotide sequence encoding an enzyme that is resistant to an inhibitor and comprises a plurality of amino acid substitutions relative to the enzyme encoded by the first polynucleotide sequence.   
     
     
         49 . The method of  claim 46 ,  47  or  48  further comprising isolating the further polynucleotide sequence. 
     
     
         50 . The method of  claim 46 ,  47  or  48  wherein in step b) a mixture of the selected isolated polynucleotides of step a) are mutated. 
     
     
         51 . The method of  claim 48  wherein the further polynucleotide sequence encodes an enzyme that has increased thermostability relative to the first polynucleotide sequence. 
     
     
         52 . The method of  claim 47  wherein the property is specific activity, enzyme activity, catalytic turnover, Km or substrate utilization. 
     
     
         53 . The method of  claim 46  wherein the property is specific activity, enzyme activity, signal stability, Km or substrate utilization. 
     
     
         54 . The method of  claim 46  wherein the property is resistance to an inhibitor. 
     
     
         55 . The method of  claim 47  or  48  wherein the enzyme is DNA polymerase or RNA polymerase. 
     
     
         56 . The method of  claim 47  or  54  wherein the enzyme is resistant to inhibition by a substrate analog of the enzyme. 
     
     
         57 . The method of  claim 46  or  48  wherein the mutating employs oligonucleotides having at least one codon encoding a consensus amino acid. 
     
     
         58 . The method of  claim 47  where the enzyme is detectable in crude cellular lysates or cells. 
     
     
         59 . The method of  claim 58  wherein the enzyme is luciferase, chloramphenicol acetyltransferase, beta-glucuronidase or beta-galactosidase. 
     
     
         60 . The method of  claim 46  wherein the luciferase is a beetle luciferase. 
     
     
         61 . The method of  claim 48  wherein the first polynucleotide sequence encodes LucPpe2. 
     
     
         62 . The method of  claim 48  wherein the first polynucleotide sequence encodes LucPpl. 
     
     
         63 . The method of  claim 47  or  48  wherein the enzyme is a luciferase. 
     
     
         64 . The method of  claim 63  wherein the luciferase is a beetle luciferase. 
     
     
         65 . The method of  claim 63  wherein the first polynucleotide sequence encodes LucPpe2. 
     
     
         66 . The method of  claim 63  wherein the first polynucleotide sequence encodes LucPpl. 
     
     
         67 . The method of  claim 46 ,  47  or  48  wherein at least one amino acid substitution is to a consensus amino acid. 
     
     
         68 . The method of  claim 46 ,  47  or  48  wherein the first polynucleotide sequence is subjected to recombination mutagenesis. 
     
     
         69 . The method of  claim 46 ,  47  or  48  wherein the first polynucleotide sequence is subjected to point mutagenesis. 
     
     
         70 . The method of  claim 46 ,  47  or  48  wherein the selection is an automated multi-parameter process. 
     
     
         71 . The method of  claim 46  or  63  wherein the luciferase has increased luminescence intensity, increased signal stability or decreased Km. 
     
     
         72 . A polynucleotide sequence which is obtained by the method of  claim 46 ,  47  or  48 . 
     
     
         73 . A fusion protein comprising the luciferase of  claim 20 . 
     
     
         74 . A fusion protein comprising the luciferase of  claim 21 . 
     
     
         75 . An isolated nucleic acid molecule comprising a nucleic acid sequence encoding a thermostable beetle luciferase that retains at least 50% activity in aqueous solution for at least 2 hours at a temperature of about 50° C., wherein the thermostable beetle luciferase has a plurality of amino acid substitutions at residues corresponding to residue 2, 27, 91, 144, 173, 183, 204, 220, 221, 262, 294, 353, 354, 356, 386, 394, 399, 412, 413 or 499 of SEQ ID NO:37, wherein the plurality of substitutions is associated with enhanced thermostability relative to a corresponding beetle luciferase without the plurality of substitutions. 
     
     
         76 . The isolated nucleic acid molecule of  claim 75  wherein the thermostable beetle luciferase retains activity for at least 100 days at a temperature of about 22° C. 
     
     
         77 . The isolated nucleic acid molecule of  claim 75  wherein the thermostable beetle luciferase retains activity for at least 12 hours at a temperature of about 37° C. 
     
     
         78 . The isolated nucleic acid molecule of  claim 75  wherein the thermostable beetle luciferase retains activity for at least 2 hours at a temperature of about 65° C. 
     
     
         79 . The isolated nucleic acid molecule of  claim 75  wherein the thermostable beetle luciferase retains activity for at least 5 hours at a temperature of about 50° C. 
     
     
         80 . The isolated nucleic acid molecule of  claim 75  wherein the thermostable beetle luciferase retains activity for at least 10 hours at a temperature of about 50° C. 
     
     
         81 . The isolated nucleic acid molecule of  claim 75  wherein the thermostable beetle luciferase retains activity for at least 2 hours at a temperature of about 60° C. 
     
     
         82 . The isolated nucleic acid molecule of  claim 75  wherein the thermostable beetle luciferase retains activity for at least 5 hours at a temperature of about 60° C. 
     
     
         83 . The isolated nucleic acid molecule of  claim 75  wherein the thermostable beetle luciferase retains activity for at least 24 hours at a temperature of about 37° C. 
     
     
         84 . The isolated nucleic acid molecule of  claim 75  wherein the thermostable beetle luciferase retains activity for at least 2 days at a temperature of about 37° C. 
     
     
         85 . The isolated nucleic acid molecule of  claim 75  wherein the thermostable beetle luciferase retains activity for at least 200 days at a temperature of about 22° C. 
     
     
         86 . The isolated nucleic acid molecule of  claim 75  wherein the thermostable beetle luciferase retains activity for at least 500 days at a temperature of about 22° C. 
     
     
         87 . The isolated nucleic acid molecule of  claim 75  wherein the thermostable beetle luciferase retains activity for at least 800 days at a temperature of about 22° C. 
     
     
         88 . The isolated nucleic acid molecule of  claim 75  wherein the thermostable beetle luciferase retains activity for at least 6 hours at a temperature of about 65° C. 
     
     
         89 . The isolated nucleic acid molecule of  claim 75  wherein the thermostable beetle luciferase retains activity for at least 2 days at a temperature of about 65° C. 
     
     
         90 . The isolated nucleic acid molecule of  claim 75  wherein the thermostable beetle luciferase retains at least 90% activity for at least 40 days at a temperature of about 22° C. 
     
     
         91 . The isolated nucleic acid molecule of  claim 75  wherein at least one substitution in the thermostable beetle luciferase is at a consensus site. 
     
     
         92 . The isolated nucleic acid molecule of  claim 75  wherein at least one substitution in the thermostable beetle luciferase is to a consensus amino acid. 
     
     
         93 . The isolated nucleic acid molecule of  claim 75  wherein the thermostable beetle luciferase has a plurality of the amino acid substitutions of SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:44, SEQ ID NO:47, or SEQ ID NO:45. 
     
     
         94 . The isolated nucleic acid molecule of  claim 75  wherein the thermostable beetle luciferase has a plurality of the amino acid substitutions of SEQ ID NO:14, SEQ ID NO:19, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:44, SEQ ID NO:47, or SEQ ID NO:45. 
     
     
         95 . The isolated nucleic acid molecule of  claim 75  wherein the thermostable beetle luciferase further comprises increased luminescence intensity or signal stability relative to the corresponding beetle luciferase. 
     
     
         96 . The isolated nucleic acid molecule of  claim 75  wherein the thermostable beetle luciferase further comprises a decreased Km. 
     
     
         97 . A kit comprising: a container comprising the isolated nucleic acid molecule of  claim 75 . 
     
     
         98 . An isolated nucleic acid molecule encoding a thermostable beetle luciferase that retains at least 50% activity in aqueous solution for at least 2 hours at a temperature of about 50° C., wherein the thermostable beetle luciferase has a plurality of amino acid substitutions relative to a corresponding beetle luciferase which does not retain at least 50% activity in aqueous solution for at least 2 hours at a temperature of about 50° C., wherein at least three substitutions are associated with increased thermostability, and wherein at least one substitution is to a consensus amino acid identified by comparing a plurality of wild-type beetle luciferases selected from the group consisting of  Luciola cruciata  luciferase,  Luciola lateralis  luciferase,  Pyrophorus plagiophthalamus  luciferase,  Pyrocoelia miyako  luciferase,  Lampyris noctiluca  luciferase,  Photuris pennsylvanica  luciferase,  Phengodes  sp. luciferase and  Photinus pyralis  luciferase. 
     
     
         99 . The isolated nucleic acid molecule of  claim 98  wherein the corresponding beetle luciferase has SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, or SEQ ID NO:37.