The invention relates to aequorin and obelin mutants whose emission is shifted with respect to wild type. The shift in emission is accomplished using a combination of mutations of amino acids within aequorin or obelin that affect bioluminescence; use of different types of chromophores, i.e., coelenterazines with variable emission characteristics; and modifications of the photoprotein with fluorophores that will allow for emission of light at longer wavelengths as a result of energy transfer. Additionally, an assay employing aequorin mutants to test for HIV-1 protease inhibitors is disclosed

Daunert, Sylvia

Deo, Sapna K.

Dikici, Emre

Rowe, Laura

English

University of Kentucky

University of KentuckyUKnowledgeChemistry Faculty Patents Chemistry3-18-2008Aequorin and Obelin Mutants with DifferingWavelengths and BioluminescenceSylvia DaunertUniversity of Kentucky, daunert@uky.eduSapna K. DeoUniversity of KentuckyEmre DikiciUniversity of KentuckyLaura RoweUniversity of Kentucky, laura.rowe@uky.eduRight click to open a feedback form in a new tab to let us know how this document benefits you.Follow this and additional works at: https://uknowledge.uky.edu/chemistry_patentsPart of the Chemistry CommonsThis Patent is brought to you for free and open access by the Chemistry at UKnowledge. It has been accepted for inclusion in Chemistry Faculty Patentsby an authorized administrator of UKnowledge. For more information, please contact UKnowledge@lsv.uky.edu.Recommended CitationDaunert, Sylvia; Deo, Sapna K.; Dikici, Emre; and Rowe, Laura, "Aequorin and Obelin Mutants with Differing Wavelengths andBioluminescence" (2008). Chemistry Faculty Patents. 13.https://uknowledge.uky.edu/chemistry_patents/13US007345160B2 (12) United States Patent Daunert et a]. US 7,345,160 B2 Mar. 18, 2008 (10) Patent N0.: (45) Date of Patent: (54) (75) (73) (*) (21) (22) (65) (51) (52) (58) (56) AEQUORIN AND OBELIN MUTANTS WITH DIFFERING WAVELENGTHS AND BIOLUMINESCENCE Inventors: Assignee: Notice: Appl. No.: Filed: Sylvia Daunert, Lexington, KY (US); Sapna Kamlakar Deo, Lexington, KY (US); Emre Dikici, Lexington, KY (US); Laura Rowe, Lexington, KY (Us) University of Kentucky Research Foundation, Lexington, KY (US) Subject to any disclaimer, the term of this patent is extended or adjusted under 35 U.S.C. 154(b) by 391 days. 10/811,138 Mar. 29, 2004 Prior Publication Data US 2005/0214776 A1 Sep. 29, 2005 Int. Cl. C07H 21/04 (2006.01) C12P 21/02 (2006.01) C07K 14/435 (2006.01) US. Cl. ................... .. 536/235; 435/69.1; 530/350 Field of Classi?cation Search ............. .. 536/23.5; 435/691; 530/350 See application ?le for complete search history. References Cited U.S. PATENT DOCUMENTS 5,023,181 A 5,093,240 A 5,139,937 A 5,162,227 A 6/1991 3/1992 8/1992 11/1992 Inouye Inouye et a1. Inouye et a1. Cormier 5,360,728 5,422,266 5,491,084 5,541,309 5,744,579 5,766,941 11/1994 6/1995 2/1996 7/1996 4/1998 6/1998 Prasher Cormier et a1. Chal?e et a1. Prasher Cormier et a1. Cormier et a1. 5,798,441 8/1998 Cormier et a1. 5,876,995 3/1999 Bryan ...................... .. 435/189 FOREIGN PATENT DOCUMENTS 0 226 979 A2 7/1987 0 245 093 A1 11/1987 0 245 093 B1 11/1987 0 540 064 A1 5/1993 OTHER PUBLICATIONS Current Protocols in Molecular Biology, Ausubel et al., eds, Unit 6.3, John Wiley & Sons, Inc., Hoboken, New Jersey, 1993, printed from the Internet on Apr. 23, 2007.* (Continued) EP EP EP EP Primary ExamineriRebecca E. Prouty Assistant ExamineriRosanne Kosson (74) Attorney, Agent, or F irmiMcDermott Will & Emery LLP (57) ABSTRACT The invention relates to aequorin and obelin mutants Whose emission is shifted With respect to Wild type. The shift in emission is accomplished using a combination of mutations of amino acids Within aequorin or obelin that affect biolu minescence; use of different types of chromophores, i.e., coelenteraZines with variable emission characteristics; and modi?cations of the photoprotein with ?uorophores that Will allow for emission of light at longer Wavelengths as a result of energy transfer. Additionally, an assay employing aequorin mutants to test for HIV-1 protease inhibitors is disclosed. 2 Claims, 18 Drawing Sheets a table showing the emission wavelength maximum (nm) of aequorin mutants with coelenterazine analogues. Coelenterazine Wild Type Aequorin Aequorin Aequorin Analogue Aequorin Mutant S Mutant S Mutant S (Aeq3) SerSCys (Aeq5) Tyrl321le CTZ i 472 491 491 487 CTZ ip 472 470 454 453 CTZ h 472 476 471 471 CTZ hep 472 476 448 465 CTZ cp 472 470 456 457 CTZ fcp 472 466 471 471 CTZ f 472 490 473 471 CTZ n 472 487 CTZ native 472 474 471 471 * All values, except wild type aequorin, were calculated from the average of 3 trials (wild type with 2). All mutants were, except Aeq5 puri?ed to >95% purity. CTZ analogues diluted to 100 micrograms/n11 methanol. US 7,345,160 B2 Page 2 OTHER PUBLICATIONS Shimomura, O., et al. “Light-emitting properties of recombinant semi-synthetic aequorins and recombinant ?uorescein-conjugated aequorin for measuring cellular calcium” Cell Calcium (1993) 14, pp. 373-378. Kurose, K., et al. “Bioluminesence of the Cab-binding photoprotein aequorin after cysteine modi?cation” Proc. Natl. Acad. Sci. USA, Jan. 1989, vol. 86, pp. 80-84. Lewis, J.C., et al. “Bioluminescence and Secondary Structure Properties of Aequorin Mutants Produced for Site-Speci?c Conju gation and Immobilization” Bioconjugate Chem, 2000, American Chemical Society, 11, pp. 65-70. Shrestha, S., et al. “Cysteine-Free Mutant of Aequorin as a Photolabel in Immunoassay Development” Bioconjugate Chem. 2002, American Chemical Society, 13, pp. 269-275. Tsuji, Frederick L, et al. “Site-speci?c mutagenesis of the calcium binding photoprotein aequorin” Proc. Natl. Acad. Sci. USA, vol. 83, pp. 8107-8111, Nov. 1986. Prasher, Douglas, et al. “Cloning and Expression of the cDNA Coding for Aequorin, A Bioluminescent Calcium-Binding Protein,” Biochemical and Biophysical Research Communications, vol. 126, No.3, 1985, pp. 1259-1268. Prasher, Douglas C., et al. “Isolation and Expression of a cDNA Coding for Aequorin, the Cab-Activated Photoprotein from Aequorea Victoria” Methods in EnZymology, 1986, vol. 133, pp. 288-297. Prasher, Douglas C., et al., “Sequence Comparisons of Comple mentary DNAs Encoding Aequorin Isotypes” Biochemistry 1987, 26, pp. 1326-1332. Charbonneau, Harry, et al. “Amino Acid Sequence of the Calcium Dependent Photoprotein Aequorin”Biochemistry 1985, 24, pp. 6762-6771. Shimomura, Osamu et al. “Resistivity to denaturation of the apoprotein of aequorin and reconstitution of the luminescent photoprotein from the partially denaturated apoprotein” Biochem. J. (1981) 199, pp. 825-828. Inouye, Satoshi, et al. “Overexpression and Puri?cation of the Recombinant Cab-Binding Protein, Apoaequorin” J. Biochem. 105 (1989) pp. 473-477. Inouye, Satoshi, et al. “Expression of Apoaequorin Complementary DNA in Escherichia coli” Biochemistry 1986, 25, pp. 8425-8429. Inouye, Satoshi, et al. “Cloning and sequence analysis of cDNA for the luminescent protein aequorin” Proc. Natl. Acad. Sci. USA, vol. 82, pp. 3154-3158, May 1985. Prendergast, Franklyn G., et al. “Chemical and Physical Properties of Aequorin and the Green Fluorescent Protein Isolated from Aequoreaforska'loe” J. Am. Chem. Soc. vol. 17, No. 17, 1978, pp. 3448-3453. Deo, Sapna K., et al. “Bioluminescence Detection of Proteolytic Bond Cleavage by Using Recombinant Aequorin” Analytical Bio chemistry 281, pp. 87-94 (2000). Malikova, Natalia, P., et al. “Spectral tuning of obelin bioluminescence by mutations of Trp92” FEBS Letters 554 (2003) pp. 184-188. Vysotski et al. “Violet bioluminescence and fast kinetics from W92F obelin: structure-based proposals for the bioluminescence triggering and the identi?cation of the emitting species,” Biochemistry, 2003 May 27, 2003, 42(20) pp. 6013-6024. Bondar, et al. “Role of conservative residue Cys158 in the formation of an active photoprotein complex of obelin” Biochemistry (Mos cow) vol. 66, No. 9, pp. 1014-1018. Ohmiya et al. “Two excited states in aequorin bioluminescence induced by tryptophan modi?cation” FEBS, vol. 301, No. 2, Apr. 1992, pp. 197-201. Ohmiya et al. “Bioluminescence of the Cab-binding photoprotein, aequorin, after histidine modi?cation” FEBS, vol. 320, No. 3, Apr. 1993, pp. 267-270. Lewis, J.C., et al. “Bioluminescence and Secondary Structure Properties of Aequorin Mutants Produced for Site-Speci?c Conju gation and Immobilization” Bioconjugate Chem. 2000, 11, pp. 65-70. * cited by examiner U.S. Patent Mar. 18, 2008 Sheet 1 0f 18 US 7,345,160 B2 Fig. 1 Fig. 1 is a table showing the emission wavelength maximum (nm) of aequorin mutants with coelenterazine analogues. Coelenterazine Wild Type Aequorin Aequorin Aequorin Analogue Aequorin Mutant S Mutant S Mutant S (Aeq3) SerS Cys (Aeq5) Tyrl 32I1e CTZ i 472 491 491 487 CTZ ip 472 470 454 453 CTZ h 472 476 471 471 CTZ hep 472 476 448 465 CTZ cp 472 470 456 457 CTZ fcp 472 466 471 471 CTZ f 472 490 473 471 CTZ n 472 487 CTZ native 472 474 471 471 * All values, except wild type aequorin, were calculated from the average of 3 trials (wild type with 2). All mutants were, except Aeq5 puri?ed to >95% purity. CTZ analogues diluted to 100 micrograms/ml methanol. U.S. Patent Mar. 18, 2008 Sheet 2 0f 18 US 7,345,160 B2 E5 £222“;35  mg mgwm;2; NE_N6 62 >58; NB 2.2E: as_ NB2.2 8.. ._N5 ==>>Hi¢>=~zEa" 3B28%SE25N .5 -ooooom 60083 .8082 -0888 . ooooomw r oooooom (swnov) mu U.S. Patent Mar. 18, 2008 Sheet 3 0f 18 US 7,345,160 B2 an E5 5222,“;SEE ~15 - 0083. - ooooom -2500: -0883 1880a looooomw -ooooowm looooomm .looooowv (slum) ma U.S. Patent Mar. 18, 2008 Sheet 4 0f 18 US 7,345,160 B2 25 59.22258E  v .5 1 cocoon - coocom (SWHOO) mu I 882: - 0883 r oooooom U.S. Patent Mar. 18, 2008 Sheet 5 0f 18 US 7,345,160 B2 E5 522%;8  252 N63 N6 m .5 00009.. ooooom 0883 00803 coooovw (SWIIOQ) ITIH U.S. Patent Mar. 18, 2008 Sheet 6 0f 18 US 7,345,160 B2 E5 52225;8 .  @223 who@ .E I 252. - oooom - oooom 1 oooon I oooom - coco: (slum) ma - 882 - coco: ' coco: - c262. U.S. Patent Mar. 18, 2008 Sheet 7 0f 18 US 7,345,160 B2 Fig. 7 Fig. 7 is a table showing the emission wavelength maximum (nm) of aequorin mutant Mutant S Y132l, Mutant S having a 3-fluoro-1-tyrosine aequorin or a 5-fluoro-1-tyrosine non-natural amino acid in position 132 in conjunction with coelenterazine analogues CTZ i, ip, n, h, hcp, cp, fcp, f and native CTZ. Coelenterazine Wild Type Aequorin Aequorin Aequorin Analogue Aequorin Mutant S Mutant S Mutant S Tyr1321le Tyr132 Tyr132 3-?u0r0-l-tyr S-?uoro-l-trp CTZ i 472 491 51 1 495 CTZ ip 472 452 471 CTZ n 472 ' 491 500 CTZ h 472 472 498 471 CTZ hcp 472 452 471 468 CTZ cp 472 457 471 471 CTZ fcp 472 463 471 CTZ f 472 472 500 497 CTZ native 472 471 495 472 U.S. Patent Mar. 18, 2008 Sheet 8 0f 18 US 7,345,160 B2 E5 52222512 2.862 80 Q2 $522568 : $522568$588568 : 2.3622302 .862280 1/\ 3 $523568 QENEQEQSQ .. QENSQEQEOQV/ \I\/\w .5 cocoa ooooow 2503~ Qoooow oooooo oooooow U.S. Patent Mar. 18, 2008 Sheet 9 0f 18 US 7,345,160 B2 E5 5966;;0% 3o 1 0mm )2 0 oENmhmEmEoO3m 2?c3/ =2.862280 2 QENSQEQBQQ 8; 2.832280 Q8 286E280 ; 2.8522835852280 3 2.362280 , 2.855600@ .5 ooooow ooooow ooooow oooooow U.S. Patent Mar. 18, 2008 Sheet 10 0f 18 US 7,345,160 B2 A55 £922258m owncomQ3# 0‘ _Po .iilifiii‘éti . . | ON 8 % w .m. .0 I om W0 w. 5225 0I? mI cm AQB “3-2+ E52 0:22 Ill I cowmm 5:I? 2:232 563?. 55E2 ..862 .5 U.S. Patent Mar. 18,2008 Sheet 11 of 18 Fig. 11 US 7,345,160 B2 SEQ ID NO: 1 cDNA encoding wild type apoaequorin 1 aatgcaattc atctttgcat caaagaatta catcaaatct ctagttgatc aactaaattg 61 tctcgacaac aacaagcaaa cagtcaagct tacatcagac 121 ttcgacaacc caagatggat tcaatttcct tgatgtcaac 181 cacaatggaa aaat'ctctct catctgatat tgtcatcaat 241 aaccttggag caacacctga atgctgtaga agccttcttc 301 ggaggagctg gaatgaaata ctgcatatat tgaaggatgg 361 aaaaaattgg ctactgatga acgaaccaac gctcatccgt 421 atatggggtg atgctttgtt aaaatggagc cattacactg 481 gatgaatgga aagcatacac aatcatcaga agattgcgag 541 gaaacattca gagtgtgcga tcgatgttga tgagatgaca 601 agacaacatt taggattttg gcgaaaagct ctacggtgga 661 gctgtcccct aagaagctct gaagatgatg tgattttgaa 721 taaaacactg atgaattcaa gaacgatttc aatcgtttgt 781 gttgattttt gtaattagga agttgttttt ttaatcaaca 841 gaacttacaa atcgaaaaag t catgacaagc tggacgacac tgacgagatg gcaagccaaa tggtgtggaa attggagaaa tgatatcgtt caaagctgct tattgatgaa gtacacca‘tg acggtggtga tcaaaatttt acagattaaa Fig. 12 aaacaatact aagcatatgt gtctacaagg cgacacaaag actgattggc tacgccaaaa gacaaagatc ggtatcatcc agtggacaac gatcctgctt tgcaccctgg ccaaattttt tcgaatgatt SEQID NO: 2 amino acid‘ sequence for wild type apoaequorin VKLTSDFDNP RWIGRHKHMF NFLDVNHNGK ISLDEMVYKA SDIVINNLGA TPEQAKRHKD AVEAFFGGAG MKYGVETDWP AYIEGWKKLA TDELEKYAKN EPTLIRIWGD ALFDIVDKDQ NGAITLDEWK AYTKAAGIIQ SSEDCEETFR VCDIDESGQL DVDEMTRQHL GFWYTMDPAC EKLYGGAVP U.S. Patent Mar. 18,2008 Fig. 13 SEQ ID NO: 3 cDNA encoding 1 aatgcaattc atctttgcat ctagttgatc aactaaattg 61 tctcgacaac aacaagcaaa cagtcaagct tacatcagac 121 ttcgacaacc caagatggat tcaatttcct tgatgtcaac 181 ca'caatggaa aaatctctct catctgatat tgtcatcaat ' 241 aaccttggag caacacctga atgctgtaga agccttcttc 301 ggaggagctg gaatgaaata ctgcatatat tgaaggatgg 361 aaaaaattgg ctactgatga acgaaccaac gctcatccgt 421 atatggggtg atgctttgtt aaaatggagc cattacactg 481 gatgaatgga aagcatacac aatcatcaga agatagcgag 541 gaaacattca gagtgagcga tcgatgttga tgagatgaca 601 agacaacatt taggattttg gcgaaaagct Ctacggtgga 661 gctgtcccct aagaagctct gaagatgatg tgattttgaa 721 taaaacactg atgaattcaa gaacgatttc aatcgtttg't 781 gttgattttt gtaattagga agttgttttt ttaatcaaca 841 gaact-tacaa atcgaaaaag Fig. 14 Sheet 12 0f 18 US 7,345,160 B2 Mutant S apoaequorin caaagaatta catgacaagc tggacgacac tgacgagatg gcaagccaaa tggtgtggaa attggagaaa tgatatcgtt caaagctgct tattgatgaa gtacaccatg acggtggtga tcaaaatttt acagattaaa t catcaaatct aaacaatact aagcatatgt gtctacaagg cgacacaaag actgattggc tacgccaaaa gacaaagatc ggtatcatcc agtggacaac gatcctgcta tgcaccctgg ccaaattttt tcgaatgatt SEQ |D.NO: 4 amino acid sequence for "Mutant S" apoaequorin VKLTSDFDNP RWIGRHKHMF NFLDVNHNGK ISLDEMVYKA SDIVINNLGA TPEQAKRHKD AVEAFFGGAG MKYGVETDWP AYIEGWKKLA TDELEKYAKN EPTLIRIWGD ALFDIVDKDQ NGAITLDEWK AYTKAAGIIQ SSEDSEETFR . VSDIDESGQL DVDEMTRQHL GFWYTMDPAS EKLYGGAVP U.S. Patent Fig. 17. Mar. 18, 2008 Sheet 15 0f 18 Non-natural amino acids US 7,345,160 B2 Non-natural Amino Acid Analogue Representative Example Effect Fluoro-analogue .n g o O I H O NH: 3-?uoro-L-tyrosine Altered pKa and H bonding strength Amine-analogue OH 553 H=~ N p-amino-L-phenylalanine Altered electrostatic charge and H bonding strength Alkyl-analogue OH %H300 H2 p-methoxy-L-phenylalanine Altered H-bonding capability, increased bulkiness Nitro-analogue No2 OH 3; HO N 2-nitro-L-tyrosine Altered TI‘ character, altered pKa, altered H bonding strength, and steric effects Hydroxyl-analog ue HO OH iN"; 2-amino-5-hydroxy-4-methyl pentanoic acid Altered pKa, loss of n-n interactions U.S. Patent Mar. 18, 2008 Sheet 16 0f 18 US 7,345,160 B2 Fig. 18 SEQ ID NO: 5, Obelin cDNA sequence 1 acgatcgaac caaacaactc agctcacagc tactgaacaa ctcttgttgt gtacaatcaa 61 aatgtcttca aaatacgcag ttaaactcaa gactgacttt gataatccac gatggatcaa 121 aagacacaag aatggaaaaa tcaccctcga 181 tgaaattgtg ctcgaagcca caccagaaca 241 aacaaaacgc ggatgtggaa tggaatatgg 301 taaagaaatt caattggcga cttcagaact 361 caagaaatgg tggggagatg ctgtctttga 421 tattttcgac gaatggaaag cttatggaaa 481 aatctctggt acatttcgac attgcgattt 541 ggacaacagt caacatcttg gattctggta 601 cactttggac gttccctaag ctttttttcg 661 aa cacatgtttg tccaaggcat catcaagttt gccttcccac gcaagaaacg aaagatggaa atctctccat ggtgaccttg ccagaagctg atttcctcga ctgatgacat gtgttgaagc aattcctcga aacctactct gtggtacaat cacaagaaga atgttgacga atggtctcta catcaatgga atgtgccaag tttctttaga tggatggaaa cattcgtgaa cactttggac ttgtgaagcg gatgacaaga tggcaacgga U.S. Patent Mar. 18, 2008 Sheet 17 0f 18 US 7,345,160 B2 Fig. 19. SEQ ID NO: 6, Native Obelin Amino Acid Sequence MSSKYAVKLK TDFDNPRWIK RHKHMFDFLD INGNGKITLD EIVSKASDDI CAKLEATPEQ TKRHQVCVEA FFRGCGMEYG KEIAFPQFLD GWKQLATSEL KKWARNEPTL IREWGDAVFD IFDKDGSGTI TLDEWKAYGK ISGISPSQED CEATFRHCDL DNSGDLDVDE MTRQHLGFWY TLDPEADGLY GNGVP U.S. Patent Mar. 18, 2008 Sheet 18 0f 18 US 7,345,160 B2 Fig. 20 Obelin mutants with Coelenerazine analogues. Coelenterazine Analogs Emission Max (nm) Obelin i ip h hcp cp fcp f ctz Mutant C75 8- 521 478 503 478 472 497 503 491 C51 S C75S- 505 471 500 471 471 487 504 491 G678 C1588 506 472 497 478 472 475 497 491 C1-51S 497 471 490 471 471 474 493 491 

Aequorin and Obelin Mutants with Differing Wavelengths and Bioluminescence

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Aequorin and Obelin Mutants with Differing Wavelengths and Bioluminescence

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