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OE Isolated and Purified Nucleic Acid Molecules That Encode A USOO6232.107B1 (12) United States Patent (10) Patent No.: US 6,232,107 B1 Bryan et al. (45) Date of Patent: May 15, 2001 (54) LUCIFERASES, FLUORESCENT PROTEINS, 4,676,406 6/1987 Frischmann et al. ................ 222/136 NUCLEC ACDS ENCODING THE 4,681870 7/1987 Balint et al. ......................... 502/403 LUCIFERASES AND FLUORESCENT 4,714,682 12/1987 Schwartz. ... 436/10 4,717,158 1/1988 Pennisi. 273/58 A PROTEINS AND THE USE THEREOF IN 4,735,660 4/1988 Cane ........ ... 106/203 DIAGNOSTICS, HIGH THROUGHPUT 4,745,051 5/1988 Smith et al. ........................... 435/68 SCREENING AND NOVELTY TEMS 4,762,881 8/1988 Kauer ................................ 525/54.11 4,765,510 8/1988 Rende ... ... 222/79 (75) Inventors: Bruce J. Bryan, 716 N. Arden Dr., 4,767.206 8/1988 Schwartz. ... 356/73 Beverly Hills, CA (US) 90210; 4,774,189 9/1988 Schwartz. ... 436/10 Christopher Szent-Gyorgyi, Pittsburgh, 4,777,128 10/1988 Lippa ....................................... 435/5 PA (US) 4,781,647 11/1988 Doane, Jr. ............................ 446/219 4,789,633 12/1988 Huang .......... ... 435/240.2 (73) Assignees: Bruce J. Bryan; Prolume, LTD, 4.853,327 8/1989 Dattagupta ............................... 435/6 Pittsburgh, PA (US) 4,861,709 8/1989 Ulitzur et al. ........................... 435/6 4,867,908 9/1989 Recktenwald et al. .. ... 252/408.1 (*) Notice: Subject to any disclaimer, the term of this 4870,009 9/1989 Evans et al. ........................... 435/70 patent is extended or adjusted under 35 4.882,165 11/1989 Hunt et al. ... 424/450 U.S.C. 154(b) by 0 days. 4,891,043 1/1990 Zeimer et al. ... ... 604/20 4,908.405 3/1990 Bayer et al. ........................... 525/61 (21) Appl No.: 09/277,716 4,921,757 5/1990 Wheatley et al. ... ... 428/402.2 y - - - 9 4924,358 5/1990 Von Heck .............................. 362/32 (22) Filed: Mar. 26, 1999 4,927.923 5/1990 Mathis et al. 540/456 O O 4.950,588 8/1990 Dattagupta ............................... 435/6 Related U.S. Application Data 4,952,496 8/1990 Studier et al. ......................... 435/91 (60) Provisional application No. 60/102.939, filed on Oct. 1, 4,963,117 10/1990 Gualdoni .............................. 446/219 1998, provisional application No. 60/089,367, filed on Jun. 4.968,613 11/1990 Masuda et al. ... 435/172.3 15, 1998, and provisional application No. 60/079,624, filed 5,004,565 4/1991 Schaap ................................. 252/700 on Mar. 27, 1998. 5,023,181 6/1991 Inouye ................................. 435/189 (51) Int. Cl. ............................. C12N 9/02; G01N 33/53; (List continued on next page.) C12O 1/66; C12P 21/06 FOREIGN PATENT DOCUMENTS (52) U.S. Cl. ................................... 435/189; 435/6; 435/8; 3935974 5/1991 (DE). 435/69.1; 435/183; 435/252.2; 435/320.1 OO2S350 9/1980 (EP). O194102 9/1986 (EP). (58) Field of Search .................................... 435/69.1, 189, 435/252.3, 252.33, 320.1, 6, 8, 69.7, 183; (List continued on next page.) 536/23.2 OTHER PUBLICATIONS (56) References Cited Baldwin et al., Cloning of the luciferase Structural genes U.S. PATENT DOCUMENTS from Vibro harveyi and expression of bioluminescence in Escherichia coli, Biochemistry 23: 3663–3667 (1984). 2,541,851 2/1951 Wright - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - 260/37 Belas et al., Bacterial bioluminescence: Isolation and 3,384,498 5/1968 Ahrabi .......... 3,511,612 5/1970 Kennerly et al. - - - 1. expression of the luciferase genes from Vibrio harveyi, 3,565,815 2/1971 Christy ........... 252/301.3 Science 218: 791-793 (1982). 3,584.211 6/1971 Rauhut ....... - - - - - - - 24.0/2.25 Blinks et al., Multiple forms of the calcium-sensitive biolu 3,634,280 1/1972 Dean et al. 252/301.3 R minescent protein aequorin, Fed. Proc. 1435: 474 (1975). 3,649,029 3/1972 Worrell ................................ 273/186 3,661,790 5/1972 Dean et al. ........ 252/301.3 R (List continued on next page.) 3,669,891 6/1972 Greenwood et al. .................. 252/90 3,727,236 4/1973 Lloyd et al. ............................... 2.5i Primary Examiner—Ponnathapu Achutamurthy 3,873,485 3/1975 Fichera ....... 260/292 Assistant Examiner Manjunath N. Rao 4,021,364 5/1977 Speiser ... ... 252/316 (74) Attorney, Agent, or Firm-Stephanie Seidman; Heller, 4.175,183 11/1979 Ayers ..................................... 536/57 Ehrman, White & Mculiffe LLP 4,177,038 12/1979 Biebricher et al. ... 8/192 4,225,581 9/1980 Kreuter et al. ......................... 424/88 (57) ABSTRACT 5. "E. E." "...O.E. Isolated and purified nucleic acid molecules that encode a 4,282,287 8/1981 Giese ......... ... 428/407 luciferase from Renilla mulleri, Gaussia and Pleuromamma, 4,313.843 2/1982 Bollyky et al. 252/188.3 and the proteins encoded thereby are provided. Isolated and 4,324,683 4/1982 Lim et al. .......... ... 252/316 purified nucleic acids encoding green fluorescent proteins 4,478,817 10/1984 Campbell et al. .................... 424/7.1 from the genus Renilla and Ptilosarcus, and the green 4,528,180 7/1985 Schaeffer ............................... 424/52 fluorescent proteins encoded thereby are also provided. 4,534,317 8/1985 Walsh ................ ... 119/51 R Compositions and combinations comprising the green fluo 4,542,102 9/1985 Dattagupta et al. ..................... 435/6 rescent proteins and/or the luciferase are further provided. 4,562,157 12/1985 Lowe et al. ........... ... 435/291 4,563,726 1/1986 Newcomb et al. .................... 362/34 4,581,335 4/1986 Baldwin ............................ 435/172.3 63 Claims, 11 Drawing Sheets US 6,232,107 B1 Page 2 U.S. PATENT DOCUMENTS FOREIGN PATENT DOCUMENTS 5,093,240 3/1992 Inouye et al. ....................... 435/69.1 O226979 7/1987 (EP). 5,096,807 3/1992 Leaback ................................... 435/6 O245093 11/1987 (EP). 5,098,828 3/1992 Geiger et al. .. ... 435/7.72 O246174 11/1987 (EP). 5,139.937 8/1992 Inouye et al. .. ... 435/69.1 O386691 9/1990 (EP) ................................ C12O/1/68 5,158,349 10/1992 Holland et al. ........................ 362/34 O387355 9/1990 (EP). 5,162,227 11/1992 Cormier ...... ... 435/252.33 O194102 10/1991 (EP). 5,162,508 11/1992 Lehn et al. ............................... 401A4 O540064 5/1993 (EP). 5,169,784 12/1992 Summers et al. 435/320.1 O713O89 5/1996 (EP). 5,171,081 12/1992 Pita et al. ............................... 362/34 2292595 6/1976 (FR). 5,182,202 1/1993 Kajiyama et al. ................... 435/189 2288232 10/1995 (GB). 5,189,029 2/1993 Boyer et al. ... ... 514/64 63-317079 12/1988 (JP). 5,196,318 3/1993 Baldwin et al. ... ... 435/69.1 3O3O678 2/1991 (JP). 5,196.524 3/1993 Gustafson et al. ... 536/23.2 4258288 9/1992 (JP). 5,219,737 6/1993 Kajiyama et al. ... 435/69.1 5064583 3/1993 (JP). 5,221,623 6/1993 Legocki et al. 435/252.3 T222590 8/1995 (JP). 5.222,797 6/1993 Holland .................................. 362/34 7241.192 9/1995 (JP). 5,229.285 7/1993 Kajiyama et al. ................... 435/189 8603840 7/1986 (WO) .......................... GO1N/33/545 5,246,834 9/1993 Tsuji et al. ... ... 435/7.91 8703304 6/1987 (WO). 5,268,463 12/1993 Jefferson ........ ... 536/23.7 9001542 2/1990 (WO). 5,277.913 1/1994 Thompson et al. ... 424/450 92O1225 1/1992 (WO). 5,279,943 1/1994 Mathis et al. ...... ... 435/7.32 92O4577 3/1992 (WO). 5,292,658 3/1994 Cormier et al. 435/252.33 92.15673 9/1992 (WO). 5,310,421 5/1994 Shapero et al. ...................... 106/208 93.13395 7/1993 (WO). 5,323,492 6/1994 DeMars ............................... 2/203.13 9418342 8/1994 (WO). 5.330,906 7/1994 Kajiyama et al. ... 435/189 942.5855 11/1994 (WO). 5,337.745 8/1994 Benaron ............. 128/633 9507463 3/1995 (WO). 5,352,598 10/1994 Kajiyama et al. ... 435/189 9518853 7/1995 (WO) .............................. C12N/O/15 5,360,726 11/1994 Raikhel ....... ... 435/172.3 9521.191 8/1995 (WO). 5,360,728 11/1994 Prasher ................................. 435/189 952.5798 9/1995 (WO). 5,362,865 11/1994 Austin ................................. 536/24.1 96O71OO 3/1996 (WO). 5,364,797 11/1994 Olson et al. ......................... 436/501 96O7917 3/1996 (WO). 5,366,881 11/1994 Singh et al. ... 435/177 9623810 8/1996 (WO). 5,374,534 12/1994 Zomer et al. ............................ 435/8 9627675 9/1996 (WO). 5,383,100 1/1995 Kikos ..................................... 362/34 9711094 3/1997 (WO). 5,387,526 2/1995 Garner et al. 436/169 9726333 7/1997 (WO). 5,405,905 4/1995 Darr ............ 524/420 9728261 8/1997 (WO). 5,405,958 4/1995 VanGermert ... ... 544/71 972931.9 8/1997 (WO). 5,412,085 5/1995 Allen et al. 536/24.1 9741228 11/1997 (WO). 5,413,098 5/1995 Benaron ...... 128/633 98.14605 4/1998 (WO). 5,413,332 5/1995 Montgomery .......................... 273/58 9826277 6/1998 (WO) ............................ GO1 N/21/76 5,415,151 5/1995 Fusi et al. .............................. 124/56 9949019 9/1999 (WO). 5,418,155 5/1995 Cormier et al. 435/189 5,422,075 6/1995 Saito et al. ............................. 422/52 OTHER PUBLICATIONS 5,422.266 6/1995 Cormier et al. ... 435/252.3 5,424,216 6/1995 Nagano et al. ... 436/116 Casper et al. Expression of the green fluorescent protein-en 5,432,081 7/1995 Jefferson ..... ... 435/252.3 coding gene from a tobacco mosaic virus-based vector Gene 5,433,896 7/1995 Kang et al. .......................... 252/700 173: 69–73 (1996). 5,451,347SE, 9/1995E.
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