Back to results
US 2003/0068801 A1Published application
Thermostable luciferases and methods of production
AssigneePromega Corporation
InventorsKeith V. Wood, Mary P. Hall
Life of this patent
Estimating the term…
Abstract
Luciferase enzymes with greatly increased thermostability, e.g., at least half lifes 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.
Claims22
A second beetle luciferase with increased thermostability as compared with a first luciferase, said second luciferase made by the following method:
- a) mutating a polynucleotide sequence encoding the first luciferase to obtain a polynucleotide sequence encoding the second luciferase;
- b) selecting the second luciferase if a plurality of characteristics including thermostability of a luciferase is in a preferred range.
A beetle luciferase that is thermostabile for at least 2 hours at 50° C. in aqueous solution.
A method for preparing a beetle luciferase with increased thermostability, said method comprising the following steps:
- a) mutating a polynucleotide sequence encoding a first luciferase to obtain a sequence encoding a second luciferase;
- and
- b) selecting the second luciferase if a plurality of characteristics including thermostability of a luciferase are in a preferred range.
A DNA molecule having a nucleotide sequence that encodes a mutant luciferase with increased thermostablility as compared to the thermostability of a native luciferase.
A kit comprising a beetle luciferase with a half-life of at least 2 hours at 50° C.
A luciferase having an amino acid sequence consisting of
- 19 a) DPMEDKNILYGPEPFYPLADGTAGEQMFYALSRYADISGCIALTNAHTKENVLYEEFLKL SCRLAESFKKYGLKQNDTIAVCSENGLQFFLPIIASLYLGIIAAPVSDKYIERLIHSLG IVKPRIIFCSKNTFQKVLNVKSKLKYVETIIILDLNEDLGGYQCLNNFISQNSDINLDVK KFKPYSFNPDDQVLVMFSSGTTGVSKGVMLTHKNIVARFSLAKDPTFGNAINPTTAILT VIPFHHGFGMMTTLGYFTCGFRVVLMHTFEEKLFLQSLQDYKVESTLLVPTLMAFLAKSA LVEKYDLSHLKEIASGGAPLSKEIGEMVKKRFKLNFVRQGYGLTETTSAVLITPNNDVRP GSTGKIVPFHAVKVVDPTTGKILGPNETGELYFKGDMIMGYYNNEEATKAIINKDGWLR SGDIAYYDNDGHFYIVDRLKSLIKYKGYQVAPAEIEGILLQHPYIVDAGVTGIPDEAAGE LPAAGVVVQTGKYLNEQIVQNFVSSQVSTAKWLRGGVKFLDEIPKGSTGKIDRKVLRQMF EKHTNG b) DPMEDKNILYGPEPFYPLADGTAGEQMFYALSRYADISGCIALTNAHTKENVLYEELLKL SCRLESFKKYGLKQNDTIAVCSENGLQFFLPIIASLYLGIIAAPVSDKYIERELIHSLG IVKPRIIFCSKNTFQKVLNVKSKLKYVETIIILDLNEDLGGYQCLNNFISQNSDINLDVK KFKPYSFNRDDQVALVMFSSGTTGVSKGVMLTHKNIVARFSHAKDPTFGNAINPTTAILT VIPFHHGFGMMTTLGYFTCGFRVVLMHTFEEKLFLQSLQDYKVESTLLVPTLMAFFAKSA LVEKYDLSHLKEIASGGAPLSKEIGEMVRFKLNFVRQGYGLTETTSAVLITPNNDVRP GSTGKIVPFHAVKVVDPTTGKILGPNETGELYFKGDMIMKGYYNNEEATKAIINKDGWLR SGDIAYYDNDGHFYIVDRLKSLIKKGYQVAPAEIEGILLQHPYIVDAGVTGIPDEAAGE LPAAGVVVQTGKYLNEQIVQNFVSSQVSTAKWLRGGVKFLDEIPKGSTGKIDRKVLRQMF EKHTNG c) DPMEDKNILYGPEPFYPLADGTAGEQMFYALSRYADSGCIALTNAHTKENVLYEEFLKL SCRLAESFKKYGLKQNDTIAVCSENGLQFFLPIIASLYLGIIAAPVSDKYIERELIHSLG IVKPRIIFCSKNTFQKVLNVKSKLKYVETIIILDLNEDLGGYQCLNNFISQNSDINLDVK KFKPYSFNPDDQVALVMFSSGTTGVSKGVMLTHKNIVVRFSLAKDPTFGNAINPTTAILT VIPFHHGFGMMTTLGYFTCGFRVVMHTFEEKLFLQSLQDYKVESTLLVPTLMAFFAKSA LVEKYDLSHLKEIASGGAPLSKEIGEMVKKRFKLNFVRQGYGLTETTSAVLITPNNDVRP GSTGKIVPFHAVKVVDPTTGKILGPNETGELYFKGDMIMKGYYNNEEATKAIITKDGWLR SGDIAYYDNDGHFYIVDRLKSLIKYKGYQVAPAEIEGILLQHPYIVDAGVTGIPDEAAGE LPAAGVVVQTGKYLNEQIVQNFVSSQVSTAKWLRGGVKFLDEIPKGSTGKIDRKVLRQMF EKHTNG d) DPMEDKNILYGPEPFYPLADGTAGEQMFYALSRYADISGCIALTNAHTKENVLYEEFLKL SCRLAESFKKYGLKQNDTIAVCSENGLQFFLPIIASLYLGIIAAPVSDKYIERELIHSLG IVKPRIIFCSKNTFQKVLNVKSKLKYVETIIILDLNEDLGGYQCLNNFISQNSDINLDVK KFKPYSFNRDDQVALVMFSSGTTGVSKGVNLTHXNIVARFSIAKDPTFGNAINPTTAILT VIPFHHGFGMMTTLGYFTCGFRVVLMHTFEEKLFLQSLQDYKVESTLLVPTLMAFLAKSA LVEKYDLSHLKEIASGGAPLSKEIGEMVKKRFKLNFVRQGYGLTETTSAVLITPNNDVRP GSTGKIVPFHAVKVVDPTTGKILGPNETGELYFKGDMIMKGYYNNEEATKAIINKDGWLR SGDIAYYDNDGHFYIVDRLKSLIKYKGYQVAPAEIEGILLQHPYIVDAGVTGIPDEAAGE LPAAGVVVQTGKYLNEQIVQNFVSSQVSTAKWLRGGVKFLDEIPKGSTGKIDRKVLRQMF EKHTNG e) DPMEDKNILYGPEPFYPLADGTAGEQMFDALSRYADISGCIALTNAHTKENVLYEEFLKL SCRLAESFKKYGLKQNDTIAVCSENGLQFFLPIIASLYLGIIAAPVSDKYIERELIHSLG IVKPRIIFCSKNTFQKVLNVKSKLKYVETIIILDLNEDLGGYQCLNNFISQNSDINLDVK KFKPYSFNRDDQVALVMFSSGTTGVSKGVMLTHKNIVARFSHAKDPTFGNAINPTTAILT VIPFHHGFGMTTLGYFTCGFRVVLMHTFEEKLFLQSLQDYKVESTLLVPTLMAFFAKSA LVEKYDLSHLKEIASGGAPLSKEIGEMVKKRFKLNFVRQGYGLTETTSAVLITPNNDVRP GSTGKIVPFHAVKVVDPTTGKILGPNETGELYFKGDMIMKGYYNNEEATKAIINKDGWLR SGDIAYYDNDGHFYIVDRLKSLIKYKGYQVAPAEIEGILLQHPYIVDAGVTGIPDEAAGE LPAAGVVVQTGKYLNEQIVQNFVSSQVSTAKWLRGGVKFLDEIPKGSTGKIDRKVLRQMF EKHTNG f) DPMADKNILYGPEPFYPLADGTAGEQMFDALSRYADISGCIALTNAHTKENVLYEEFLKL SCRLAESFKKYGLKQNDTIAVCSENGLQFFLPVIASLYLGIIAAPVSDKYIERELIHSLG IVKPRIIFCSKNTFQKVLNVKSKLKSVETIIILDLNEDLGGYQCLNNFISQNSDINLDVK KFKPYSFNRDDQVALVMFSSGTTGVSKGLTHKNIVARFSLAKDPTFGNAINPTTAILT VIPFHHGFGMMTTLGYFTCGFRVVLMHTFEEKIFLQSLQDYKVESTLLVPTLMAFLAKSA LVEKYDLSELKEIASGGAPLSKEIGEMVKKRFKLNFVRQGYGLTETTSAVLITPKxxARPG STGKIVPFHAVKVVDPTTGKILGPNEPGELYFKGAMIMKGYYNNEEATKAIIDNDGWLRS GDIAYYDNDGHFYIVDRLKSLIKYKGYQVAPAEIEGILLQHPYIVDAGVTGIPDEAAGEL PAAGVVVQTGKYLNEQIVQDFVSSQVSTAKWLRGGVKFLDEIPKGSTGKIDRKVLRQMFE KHTNG$ g) DPMADKNILYGPEPFYPLADGTAGEQHFYALSRYADISGCIALTNAHTKENVLYEEFLKL SCRLAESFKKYGLKQNDTIAVCSENGLQFFLPVIASLYLGIIAAPVSDKYIERELIHSLG IVPRIIFCSKNTFQKVLNVKSKLKYVETIIILDLNEDLGGYQCLNNFISQNSDINLDVK KFKPYSFNRDDQVALFSSGTTGVKGVMLTHKNIVARFSLAKDPTFGNAINPTTAILT VIPFHHGFGMMTTLGYFTCGFRVVLMHTFEEKLFLQSLQDYKESTLLVPTLMAFLAKSA LVEKYDLSHLKEIASGGAPLSKEIGEMVKKRFKLNFVRQGYGLTETTSAVLITPKxxVRPG STGKIVPFHAVKVDPTTGKILGPNEPGELYFKGDMIMKGYYNNEEATKAIIDKDGWLRS GDIAYYDNDGHFYIVDRLKSLIKYKGYQVAPAEIEGILLQHPYIVDAGVTGIPDEAAGEL PAAGVVVQTGKYLNEQIVQNFVSSQVSTAKWLRGGVKFLDEIPKGSTGKIDRKVLRQMFE KHTNG h) DPMADKNILYGPEPFYPLADGTAGEQMFDALSRYADIPGCIALTNAHTKENVLYEEFLKL SCRLAESFKKYGLKQNDTIAVCSENGLQYFLPVIASLYLGIIAAPVSDKYIERELIHSLG IVPRIIFCSKNTFQKVLNVKSKLKYVETIIILDLNEDLGGYQCLNNFISQNSDINLDVK KFKPNSFNPDDQVALVMFSSGTTGVPKGVMLTHKNIVAPFSIAKDPTFGNAINPTTAILT VIPFHHGFGMMTTLGYFTCGFRVVLMHTFEEKLFLQSLQDYKVESTLLVPTLMAFLAKSA LVEKYDLSHLKEIASGGAPLSKEIGEMVKKRFKLNFVRQGYGLTETTSAVLITPKxxARPG STGKIVPFHAVKVVDPTTGKILGPNEPGELYFKGAMIMKGYYNNEEATKAIIDKDGWLRS GDIAYYDNDGHFYIVDRLKSLIKYKGYQVAPAEIEGILLQHPYIVDAGVTGIPDEAAGEL PAAGVVVQTGKYLNEQIVQNFVSSQVSTAKWLRGGVKFLDEIPKGSTGKIDRKVLRQMFE KHTNG i) DPMADKNILYGPEPFYPLADGTAGEQMFDALSRYADIPGCIALTNAHTKENVLYEEFLKL SCRLAESFKKYGLKQNDTIAVCSENGLQFFLPVIASLYLGIIAAPVSDKYVERELIHSLG IVKPRIIFCSKNTFQKVLNVKSKLKYVETIIILDLNEDLGGYQCLNNFISQNSDSNLDVK KFKPNSFNRDDQVALVMFSSGTTGVPKMLTHNIVARFSLAKDPTFGNAINPTTAILT VIPFHHGFGMMTTLGYFTCGFRVVLMHTFEEKLFLQSLQDYKVESTLLVPTLMAFLAKSA LVEKYDLSHLKEIASGGAPLSKEIGEMVKKRFKLNFVRQGYGLTETTSAVLITPKxxARPG STGKIVPFAVKVVDPTTGKILGPNEPGELYFKGAMIMKGYYNNEATKAIIDKDGWLRS GDIAYYDNDGHFYIVDRLKSLIKYKGYQVAPAEIEGILLQHPYIVDAGVTGIPDEAAGEL PAAGVVVQTGKYLNEQIVQNFVSSQVSTAKWRGGVKFLDEIPKGSTGKIDRKVLRQMFE KHTNG j) DPMADKNILYGPEPFYPLADGTAGEQMFDALSRYADIPGCIALTNAHTKENVLYEEFLKL SCRLAESFYGLKQNDTIAVCSENGLQFFLPVIASLYLGIIVAPVNDKYIERELIHSLG IVKPRIVFCSINTFQKVLNVKSKLKSVETIIILDLNEDLGGYQCLNNFISQNSDINLDVK KFKPYSFNRDDQVALIMFSSGTTGLPKGVMLTHKNIVARFSLAKDPTFGNAINPTTAILT VIPFHGFGMMTTLGYFTCGFRVVLTFEEKLFLQSLQDYKVESTLLVPTLMAFLAKSA LVEKYDLSHLKEIASGGAPLSKEIGEMVKKRFKLNFVRQGYGLTETTSAVLITPKxxARPG STGKIVPFHAVKVVDPTTGKILGPNEPGELYFKGPMIMGYYNNEEATKAIIDNDGWLRS GDIAYYDNDGHFYIVDRLKSLIKKGYQVAPAIEGILLQHPYIVDAGIVTGIPDEAAGEL PAAGVVQTGKYLNEQIVQDFVSSQVSTAKWLRGGVKFLDEIPKGSTGKIDRKVLRQMFE KHTNG k) DPMADKNILYGPEPFYPLADGTAGEQMFDALSRYADIPGCIALTNAHTKENVLYEEFLKL SCRLAESFYGLKQNDTIAVCSENGLQFFLPVIASLYLGIIVAPVNDKYIERELIHSLG IVKPRIVFCSINTFQKVLNVKSKLKSVETIIILDLNEDLGGYQCLNNFISQNSDINLDVK KFKPYSFNRDDQVALIMFSSGTTGLPKGVMLTHKNIVARFSLAKDPTFGNAINPTTAILT VIPFHGFGMMTTLGYFTCGFRVVLTFEEKLFLQSLQDYKVESTLLVPTLMAFLAKSA LVEKYDLSHLKEIASGGAPLSKEIGEMVKKRFKLNFVRQGYGLTETTSAVLITPKxxAKPG STGKIVPFHAVKVVDPTTGKILGPNEPGELYFKGPMIMGYYNNEEATKAIIDNDGWLRS GDIAYYDNDGHFYIVDRLKSLIKKGYQVAPAIEGILLQHPYIVDAGIVTGIPDEAAGEL PAAGVVVQTGKYLNEQIVQIVQDYVASQVSTAKWLRGGVKFLDEIPKGSTGKIDRKVLR QMFEKHTNG l) DPMEDKNILYGPEPFYPLADGTAGEQMFYALSRYADISGCIALTNAHTKENVLYEEFLKL SCRLAESFKKYGLKQNDTIAVCSENGLQFFLPLIASLYLGIIAAPVSDKYIERELIHSLG IVKPRIIFCSKNTFQKVLNVKSKLKYVETIIILDLNEDLGGYQCLNNFISONSDINLDVK KFKPNSFNRDDQVALVMFSSGTTGVSKGVMLTHKNIVARFSHCKDPTFGNAINPTTAILT VIPFHHGFGMMTTLGYFTCGFRVALMHTFEEKIFLQSLQDYKVESTLLVPTTLMAFFAKSA LVEKYDLSHLKEIASGGAPLSKEIGEMVKKRFVRQGYGLTETTSAVLITPDTDDVRP GSTGKIVPFHAVKVVDPTTGKILGPNETGELYFKGDMIMKSYYNNEEATKAIINKDGWLR SGDIAYYDNDGHFYIVDRLKSLIKYKGYQVAPAEIEGILLQHPYIVDAGVTGIPDEAAGE LPAAGVVVQTGKYLNEQIVQNFVSSQVSTAKWLRGGVKFLDEIPKGSTGKIDRKVLRQMF EKHKSKL m) DPMMKREKNVIYGPEPLHPLEDLTAGEMLFRALRKHSHLPQALVDVVGDESLSYKEFFEA TVLLAQSLHNCGYKNNDWSICAENNTRFFTPVIAAWYIGMIVAPVNESYIPDELCKVMG ISKPQIVFTTKNILNKVLEVQSRTNFIKRIIILDTVENIHGCESLPNFISRYSDGNIANF KPLHFDPVEQVAAILCSSGTTGLPKGVMQTHQNICVRLIHALDPRAGTQLIPGVTVLVYL PFFHAFGFSITLGYFMVGLRVIMFRRFDQEAFLKAIQDYEVRSVINVPSVILFLSKSPLV DKYDLSSLRELCCGAAPLAKEVAEVAAKRLNLPGTRCGFGLTESTSANIHSLRDEFKSGS LGRVTPLMAAKIADRETGKAIGPNQVGELCIKGPMVSKGYVNNVEATKEAIDDDGWLHSG DFGYYDEDEHFYVVDRYKELIKYKGSQVAPAELEEILLKNPCIRDVAVVGIPDLEAGELP SAFVVKQPGKEITAKEVYDYLAERVSHTKYLRGGVRFVDSIPRNVTGKITRKELLKQLLE KAGG n) DPMMKREKNVIYGPEFLHPLEDLTAGEMLFRALRKHSHLPQALVDVVGDESLSYKEFFEA TVLLAQSLHNCGYKMNDVVSICAENNTRFFIPVIAAWYIGMIVAPVNESYIPDELCKVMG ISKPQIVFTTKNILNKVLEVQSRTNFIKRITILDTVENIHGCESLPNFISRYSDGNIANF KPLHFDPVEQVAAILCSSGTTGLPKGVMQTHQNICVRLIHALDPRAGTQLIPGVTVLVYL PFFHAFGFSITLGYFMVGLRVIMFRRFDQEAFLKAIQDYEVRSVINVPSVILFLSKSPLV DKYDLSSLRELCCGAAPLAKEVAEVAAKRLNLPGIRCGFGLTESTSANIHSLRDEFKSGS LGRVTPLMAAKIADRETGKALGPNQVGELCIKGPMVSKGYVNNVEATKEIDDDGWLHSG DFGYYDEDEHFYVVDRYKELIKYKGSQVAPAELEEILLKNPCIRDVAVVGIPDLEAGELP SAFVVKQPGKEITAKEVYDYLAERVSHTKYLRGGVRFVDSIPRNVTGKITRKELLKQLLE KAGG
Dependencies are inferred from the claim text.