EP0247193A1 - Isolierung der für die luciferase-wirkung kodierenden sequenzen - Google Patents

Isolierung der für die luciferase-wirkung kodierenden sequenzen

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Publication number
EP0247193A1
EP0247193A1 EP19870900449 EP87900449A EP0247193A1 EP 0247193 A1 EP0247193 A1 EP 0247193A1 EP 19870900449 EP19870900449 EP 19870900449 EP 87900449 A EP87900449 A EP 87900449A EP 0247193 A1 EP0247193 A1 EP 0247193A1
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European Patent Office
Prior art keywords
dna
sequence
luciferase activity
hybrid
molecules
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EP19870900449
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English (en)
French (fr)
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Marlene Deluca Mcelroy
Donald Raymond Helinski
Keith Vernon Wood
Jeffrey Roux De Wet
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University of California Berkeley
University of California San Diego UCSD
University of California Los Angeles UCLA
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University of California Berkeley
University of California San Diego UCSD
University of California Los Angeles UCLA
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Publication of EP0247193A1 publication Critical patent/EP0247193A1/de
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    • C—CHEMISTRY; METALLURGY
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    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0069—Oxidoreductases (1.) acting on single donors with incorporation of molecular oxygen, i.e. oxygenases (1.13)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/52—Genes encoding for enzymes or proenzymes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
    • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
    • C12N9/2468—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1) acting on beta-galactose-glycoside bonds, e.g. carrageenases (3.2.1.83; 3.2.1.157); beta-agarase (3.2.1.81)
    • C12N9/2471—Beta-galactosidase (3.2.1.23), i.e. exo-(1-->4)-beta-D-galactanase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6897—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids involving reporter genes operably linked to promoters
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y113/00—Oxidoreductases acting on single donors with incorporation of molecular oxygen (oxygenases) (1.13)
    • C12Y113/12—Oxidoreductases acting on single donors with incorporation of molecular oxygen (oxygenases) (1.13) with incorporation of one atom of oxygen (internal monooxygenases or internal mixed function oxidases)(1.13.12)
    • C12Y113/12007—Photinus-luciferin 4-monooxygenase (ATP-hydrolysing) (1.13.12.7), i.e. firefly-luciferase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
    • C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
    • C12Y302/01023—Beta-galactosidase (3.2.1.23), i.e. exo-(1-->4)-beta-D-galactanase
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/531—Production of immunochemical test materials
    • G01N33/532—Production of labelled immunochemicals
    • G01N33/535—Production of labelled immunochemicals with enzyme label or co-enzymes, co-factors, enzyme inhibitors or enzyme substrates
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/573—Immunoassay; Biospecific binding assay; Materials therefor for enzymes or isoenzymes
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/61—Fusion polypeptide containing an enzyme fusion for detection (lacZ, luciferase)

Definitions

  • radiochemical-based assays exhibit high sensitivity, yet present significant handling and storage problems.
  • nonradiochemical-based assays that is, they are less sensitive but do not have the handling and storage problems of radiochemical assays.
  • bioluminescent assays theoretically offer the sensitivity of radiochemical assays but without the attendant problems; however, for several reasons they have not been widely utilized.
  • luciferase can be covalently attached to a ligand and then employed in any number of competitive binding assays.
  • bioluminescent assays employ luciferase isolated from either insects or bacteria, and luciferase from the bacterium Vibrio fischeri has recently been cloned. This bacterial luciferase consists of two different subunits. Because of its higher quantum efficiency and single subunit structure, insect luciferase is preferred over the multisubunit bacterial luciferase.
  • luciferase is not readily available and easily isolated; and, second, the enzyme is easily denatured and hence rendered inactive by the chemical reactions necessary to effect covalent attachment to a ligand.
  • FIGURE 1 shows the essential features of the plasmid, pKW101.
  • FIGURE 2 shows the production of plasmid, pKW104, from the plasmids pKW101, pKAD9, and pKW103, and the production of plasmid, pKW106, from pKW104 and p ⁇ C19.
  • FIGURE 3 shows the production of plasmid, pKW108, a cro-luc construct, from the plasmid pKJB824.11 and pKW106.
  • FIGURE 4 shows the production of plasmid, pKW109, from plasmids, pKWJB"Lac"ZI and pKW106.
  • FIGURE 5 shows a restriction map of the luciferase gene isolated from a genomic library.
  • FIGURE 6 shows a restriction map of the plasmid pJD200.
  • FIGURE 7 shows the plasmid construct, pJD201.
  • the bioluminescence assay of the subject invention entails cloning and expressing a region of DNA that encodes a single protein subunit with luciferase activity, and then combining this region with other regions of DNA in suitable expression systems to produce hybrid molecules useful in a variety of biological assays.
  • Cloning of DNA sequences that encode luciferase activity can be accomplished by constructing either a cDNA or genomic library from an organism that exhibits such activity.
  • Particularly useful for making a cDNA or genomic library is mRNA or DNA, respectively, isolated from the firefly Photinus pyralis. but it is to be anticipated that other insect species or worms that display the phenomena of bioluminescence can be suitably employed. Examples are Lampyridae, Elatecidae, or
  • a cDNA library is realized by isolating mRNA, particularly poly (A) + RNA by techniques well-known to those in the art, for example by chromotography on oligo[dT]-cellulose as described by Aviv and Leder in proceedings of the National Academy of Sciences, U.S.A. (1972, Vol. 69, No. 6: 1408 - 1412), and reverse transcribing the poly (A) + RNA into cDNA.
  • the cDNA can then be introduced into a suitable cloning vector and either transformed or transfected into procaryotic or eucaryotic cells. Any one of a number of vectors can be employed, including plasmids, cosmids, viral vectors, or hybrids thereof.
  • the general scheme for generating a cDNA library is to insert double-stranded cDNA made from mRNA into a suitable vector. This is conveniently accomplished by inserting the cDNA into a cloning site near a functional promoter and a translational initiation site on a suitable vector. This favored cloning site is present within the coding region of genes present on Escherichia coli plasmids or bacteriophages.
  • Several examples are well-known, particularly the ⁇ -lactamase gene of pBR322, the ⁇ -galactosidase (Lac Z) gene of pUC plasmids and ⁇ gtll, and the tryp genes of the tryptophan promoter plasmids.
  • cDNA results in a hybrid molecule, or fused polypeptides consisting of the NH2-terminal region of an E. coli protein covalently linked to a polypeptide and coded by the cloned cDNA fragment. If there are no "stop signals" separating the inserted cDNA fragment with the bacterial translation initiation signals, the hybrid RNA transcript is translated in the same "reading frame" as in the original mRNA. The fused molecule is then amenable to detection in cells that harbor the vector using a variety of immunochemical, or radiochemical nucleotide hybridization techniques.
  • ⁇ gtll a procaryotic expression vector described by Young and Davis in The Proceedings of the National Academy of Science (March 1983, 80:1194 - 1198), is attractive because it permits the construction and maintenance of large cDNA libraries, and ready detection of cells harboring cDNA regions with luciferase activity.
  • double-stranded cDNA produced from mRNA isolated from bioluminescent insects is inserted into the restriction endonuclease Eco RI site in the E.
  • coli Lac Z ( ⁇ -galactosidase) gene carried by ⁇ gtll.
  • This requires revealing ligation compatible nucleotide sequences on both the cDNA and gtll DNA to effect covalent bond formation between the two.
  • this can be realized by ligating Eco RI restriction endonuclease linkers to cDNA fragments and treating ⁇ gtll with Eco RI prior to inserting the cDNA into the phage.
  • ⁇ gtll containing foreign cDNA inserts is packaged in vitro to yield infectious phage particles and infected into a compatible strain of bacteria.
  • ⁇ gtll phage that harbor DNA sequences encoding luciferase activity are identified by plating recombinant phage on a lawn of E. coli and inducing the production of hybrid proteins having luciferase and ⁇ -galactosidase sequences by adding an inducer of Lac Z transcription.
  • the inducer enhances the level of hybrid protein production and therefore enhances the sensitivity of detecting cells harboring DNA sequences encoding luciferase antigenic activity. In many instances, addition of inducer will not be necessary as the level of hybrid protein production will be sufficiently great to be detected without its addition.
  • a suitable inducer is isopropyldio- ⁇ -D galactopyranoside (IPTG).
  • Lac Z transcription directs the expression of foreign DNA inserts, that is proteins with luciferase properties.
  • the latter are screened for luciferase antigenicity with antibody directed against luciferase by incubating the E. coli with antibody for a period of time sufficient to allo antibody binding to luciferase determinants. After a wash step to remove unbound antibody, bound luciferase antibody can be revealed by several techniques, one being to incubate the ⁇ gtll-infected antibody-treated lawn of E. coli with labeled-protein A of Staphylococcus aureus, or a second antibody directed against the luciferase antibody molecule.
  • the second antibody carries a suitable reporter molecule, such as radioactive atoms, or enzyme molecules.
  • radioactive and enzymatic reporter molecules are available for use with the second antibody, examples being radioactive iodine, and the enzyme peroxidase, respectively.
  • the lawn of cells is washed, and bound antibody revealed either by radioautography if radioactive atoms are used, or by the addition of a suitable chromogenic peroxidase substrate, particularly 4-chloro-l-naphthol if peroxidase is utilized.
  • the latter is dissolved in a suitable solvent, methanol being effective, and then added to an aqueous solution containing H 2 O 2 .
  • an alternate technique may be used that involves screening the cDNA library with a biotinlabeled second antibody, and subsequent formation of biotinavidin peroxidase complex that is revealed by hydrolysis of a chromogenic peroxidase substrate.
  • a suitable substrate is diaminobenzidine. This procedure is faster, requiring four hours to ascertain a positive plaque.
  • Luciferase antibody can be generated in rabbits as described by Green and McElroy in Biochemical Biophysical Acta (1956, 20:170 - 178) using purified firefly luciferase or by generating murine monoclonal antibodies against luciferase by the procedure of Kohler and Milstein as described in Nature (1975, 256:495 - 497), and Dosch and Gelfand in The Journal of Immunology (1977, 118:302 - 308).
  • ⁇ gtll cDNA inserts were isolated preparative to identifying those that encode molecules with luciferase activity in a suitable expression vector, and to construct plasmids useful to express hybrid proteins. This was accomplished by isolating plaques that exhibit luciferase antigenicity, and the recombinant phage amplified by infecting a suitable strain of E. coli DNA from recombinant ⁇ gtll phage was isolated from the desired plaque by one of several techniques, one being to isolate phage DNA from plate lysates performed as described in Maniatis, Fritsch, and Sandbrook in Molecular Cloning (1982, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, pp. 371 - 372).
  • Isolated recombinant phage DNA was then cut with Eco RI to release cDNA inserts.
  • the latter were purified preparative to inserting the cDNA fragment into a suitable expression vector.
  • Several methods can be used to purify the restriction fragment, particularly facile is the use of agarose gel electrophoresis onto a suitable paper substrate as described by Maniatis et al., supra .
  • cDNAs of various sizes were inserted into a host cell by transformation into procaryotes or transfection into eucaryotes, or into a suitable expression vector followed by transformation or by transfection. The latter procedure is more efficient and thus favored.
  • procaryotic and eucaryotic vectors or "shuttle" vectors are routinely used for this purpose.
  • the latter vectors are capable of replicating in both procaryotes and eucaryotes, as described by Struhl, Stinchcomb, Scherer, and Davis in The Proceedings of the National Academy of Sciences. U.S.A. (1979, 76:1035 - 1039).
  • a general description of expression vectors and methods of using and isolating the same can be found by Grover in DNA Cloning. Volume II, A Practical Approach (1985, IRL Press, Oxford, Washington, D.C.) or by Maniatis et al., supra.
  • cDNA inserts obtained from ⁇ gtll, or other vectors used to construct the initial gene library, that encode protein with luciferase antigenicity will be of varying size.
  • a number of different sized inserts may exhibit luciferase enzymatic activity. Regardless of the size of the insert, it can be assayed for luciferase activity and used to produce molecules with luciferase alone, or to construct hybrid molecules exhibiting luciferase activity, and antigenic and/or enzymatic activity.
  • cDNA expression is assayable by inserting the cDNA so as to come under the transcriptional and translational control mechanisms of either the vector employed and/or the host cell chosen to carry the vector. This procedure may require inserting suitable "linkers" into either the vector, the cDNA, or both.
  • suitable "linkers” into either the vector, the cDNA, or both.
  • the expression product can be tested for luciferase enzymatic activity by preparing an extract of cells containing the cDNA insert, and adding to the extract the necessary co-factors to produce bioluminescence.
  • this assay is performed in a buffered solution at near neutral pH containing magnesium chloride, luciferin, and adenosine triphosphate. Light emission was detected with a luminometer.
  • DNA sequences that encode luciferase activity by generating a cDNA library
  • genomic DNA library This is accomplished by isolating DNA from a suitable bioluminescent insect or worm, particularly P. pyralis. and then fragmenting the DNA into fragments that are up to 20 kilobases long. This can be accomplished by using one or more restriction endonucleases followed by ligating the fragments into a suitable vector at a restriction site as described for generating a cDNA library.
  • a useful restriction endonuclease is Sau3A, which yields 12 - 20 kilobase DNA fragments when the DNA is partially digested.
  • DNA restriction endonucleases allows for ready cloning of the fragments, and thus is the preferred method of fragmenting insect DNA
  • the size of the DNA fragments dictates the ease and convenience of cloning; and while there is anticipated to be a lower upper limit comprising a DNA sequence that encodes luciferase activity, it appears that a fragment in the range of about
  • Cloning of genomic DNA fragments to produce a genomic DNA library can be effected in a variety of plasmids, cosmids, viral vectors, or hybrids thereof, similar to those used to produce a cDNA library.
  • Vectors capable of replicating in either procaryotes, eucaryotes, or both, that is shuttle vectors, are similarly utilized to generate a genomic library.
  • Shuttle vectors are the preferred recipients of the DNA fragments as they permit growing the vector in usable quantities in bacteria, and then testing for the presence of the desired DNA sequence in eucaryotes. Regardless of the vectors used, all should contain one or more selectable markers.
  • DNA luciferase sequences are amplified and cells harboring DNA luciferase sequences identified. This can be achieved using immunochemical antibody detection techniques if the sequences are inserted into vectors compatible with such screening, such as pUC plasmids, ⁇ gtll, or the like. Alternatively, DNA luciferase sequences can be ascertained using tracer-labeled homologous nucleotide probes.
  • the latter can be generated by a variety of techniques well-known to those skilled in the art, particularly 32 p nick-translation techniques of cDNA-cloned probes with luciferase sequences, or by synthesizing an oligonucleotide probe homologous to luciferase DNA sequences.
  • the latter probes are derived from a knowledge of luciferase immunoacid sequences translatable into nucleotide sequences via the genetic code.
  • other less facile techniques are available for defining genomic DNA sequences such as employing labeled luciferase mRNA. Once cells are identified that harbor a recombinant vector exhibiting DNA luciferase sequences, the DNA from these structures can be isolated by techniques well-known in the art.
  • DNA inserts can be isolated from either library and used by themselves or in combination to construct expression vectors that produce hybrid molecules, particularly proteins that exhibit both luciferase activity and a second functionality. This can be accomplished by isolating the cDNA or the genomic DNA inserts and ligating either into a suitable expression vector at a site near a functional promoter and a translational initiation site on the promoter. The latter, of course, exhibits cell-selectable markers, as well as the necessary replication and regulation features associated with growing such vectors and expressing DNA inserts contained therein. Examples of such are genes that code for drug resistance, enhancers upstream activation sites (UASs), as well as transcriptional control units that are not found on the upstream, i.e., the 5', side of the genes regulated.
  • UASs enhancers upstream activation sites
  • DNA sequence can be mutated using a variety of techniques and chemicals to yield chemicals with altered luciferase activity. This can be accomplished by techniques well-known in the art, particularly those described by Smith in The Annual Review of the Genetics (1985, pp. 423 - 462). Thus, DNA can be deleted from either the 5' or the 3' ends of the molecule, or mutagens such as sodium bisulfite can be employed. Following selection of cells harboring the mutant molecules, the latter can be isolated and assayed for features not associated with native luciferase.
  • Native luciferase utilizes adenosine triphosphate, and it can be expected that the molecule exhibiting guanosine triphosphate hydrolysis specificity will be obtained. Additionally, mutant molecules that exhibit altered light emission properties can also be expected to be produced by these techniques.
  • cDNA, genomic DNA, or hybrids constructed thereof encoding luciferase activity into bacteria or eucaryotic cells by expression vectors, the same can also be inserted into plants by one of two routes.
  • a variety of vectors primarily based on Ti plasmids isolated from the bacteria Agrobacterium tumefaciens can be utilized. This involves inserting DNA sequences encoding luciferase activity downstream of a suitable plant cell promoter, transforming the bacterium with the recombinant plasmid, and then infecting plant cells with the bacterium.
  • the most commonly used plasmids are the octopine or nopaline types. This procedure is preferred for dicotyleydonous species.
  • a second method for transferring DNA sequences encoding luciferase is to electroporate vectors containing the same directly into plant cells. This procedure complements Aorobacterium invection as it permits the transformation of monocotyledonous plant species.
  • luciferase can be employed to measure the amount of biomass present in a self-propagating system as described by De Luca and McElroy in Methods in Enzymology (1978, Academic Press, New York, 57:3 - 15).
  • Hybrid molecules exhibiting luciferase activity and a second protein capable of binding to cell associated cytoplasmic or plasma membrane structures can be expected to be favorably employed in a variety of cytochemical assays.
  • hybrid molecules exhibiting luciferase activity and Protein A binding activity to detect cell surface or cytoplasmic antibody via the Protein A antibody binding portion of the molecule, and detecting the amount bound, or the cell types that exhibit binding, by assaying for bioluminescence emitted by the luciferase active portion of the molecule in the presence of its requisite substrates.
  • Construction of hybrid molecules exhibiting luciferase activity and a second biological activity permit the use of the same in a wide variety of immunochemical, cytochemical, etc., assays.
  • antibodies can be raised against the nonluciferase region of the molecule and used in standard solid or liquid phase immunoassays to detect the presence of molecules with similar antigenicity in biological fluids. In this instance, detection and quantitation of the latter would be based on the intensity of light emission from the luciferase portion of the hybrid molecules.
  • Example 1 Construction of a cDNA Library from P. pyralis.
  • total RNA was isolated from the lanterns of fireflies, P. pyralis. The tissue was homogenized in guanidinium thiocyanate, and the RNA pelleted through a cesium chloride cushion as described by Chirgwin, Przbyal, MacDonald, and Rutter in Biochemistry (1979, 18:5294 - 5298).
  • Poly (A) + RNA was isolated by chromotography on oligo(dT) -cellulose as described by Aviv and Leder in Proceedings of the National Academy of Science. U.S.A. (1972, 69:1408 - 1412).
  • the poly (A) + RNA was eluted from the column with water and then precipitated with 1/10 volume of 3 molar sodium acetate and 2.5 volumes of ethanol were added. The RNA was precipitated overnight at -20°C and pelleted. The pellet was washed with 70% ethanol, lyophilized, and the poly (A) + RNA was dissolved in water and frozen.
  • the poly (A) + RNA was used to generate cDNA as described by Maniatis, Fritsch, and Sandbrook in Molecular; Cloning (1982, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, pp. 229 - 246) with the following modifications.
  • Unlabeled deoxyribonucleoside triphosphates were present during first-strand synthesis at 200 ⁇ M, poly (A) + RNA was used at 50/_g/ml, and reverse transcriptase at 2000 units/ml (obtained from Boehringer Mannheim Biochemicals), and [ ⁇ - 32 p]dCTP at 250 ⁇ curies/ml. Additionally, second-strand cDNA was synthesized by the technique of Gubler and Hoffman in Gene (1983, 25:263 - 269).
  • This procedure employs RNase H and DNA polymerase 1, and has the advantage of generating cDNA libraries from submicrogram quantities of poly (A) + RNA as it eliminates the vector primer system and the classical hairpin loop S1 nuclease cleavage steps, which result in low cloning efficiency.
  • Nucleotides were removed after the synthesis of the first and second strands of the cDNA by ethanol precipitation and a wash with 70% ethanol.
  • doublestranded cDNA was treated with Eco RI methylase, the latter obtained from New England Biolabs.
  • Phosphorylated Eco RI linkers (pGGAATTCC) also obtained from New England Biolabs were ligated to the double-stranded cDNA in 66 ⁇ m Tris pH 7.5, 6.6 ⁇ M magnesium chloride, 10 ⁇ M dithiothreitol, and l ⁇ ATP. ⁇ gtll DNA was prepared by first ligating the cohesive ends and cutting at the unique Eco RI site. The Eco RI ends were dephosphorylated to prevent spontaneous rejoining with alkaline phosphatase using the conditions described by Maniatis, Fritsch, and Sandbrook in Molecular; Cloning (1982, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, pp. 133 - 134).
  • linker cDNA was ligated to lOjig of dephosphorylated, Eco Rl-cut ⁇ gtll with 7 Weiss units of T4 DNA ligase, also obtained from New England Biolabs.
  • the reaction was carried out for 12 hours at 4°C in a total volume of lOul.
  • the ligated ⁇ gtll phage was packaged in vitro according to the protocol of the supplier of the ⁇ -packaging kit, which was obtained from Bethesda Research Laboratories.
  • the cDNA phage library was titered on E.
  • the proportion of phage carrying cDNA inserts can be determined by plating dilutions of Y1088 in the presence of IPTG and X-g Under these conditions, ⁇ gtll without inserts produces blue plaques, whereas phage carrying cDNA produces white plaques. Using this method, it was determined that packaging of lug of ⁇ gtll DNA ligated to cDNA yields approximately 100,000 recombinant phage, and after amplification of the library on Y1088, the recombinants represented approximately 10% of the total phage population.
  • the ⁇ gtll phage library described above was plated on a lawn of E. coli strain Y1090 [ ⁇ lac V169 proA + ⁇ lon araD139 strA supF trpC22:Tn10(pMC9)], and induced with isopropyl thiol- ⁇ -D-galactopyranoside as described by Young and Davis in Science, supya.
  • the plate containing the lawn of phageinfected bacteria was overlaid with an inducer-impregnated nitrocellulose filter. Plates containing overlaid filters were incubated at 37°C for 2 to 8 hours. The position of the filter was marked with a needle so that it could later be realigned with plaques on the dish.
  • the filter was then transferred to a clean petri dish and washed with buffer containing 0.17M NaCl, 0.01M Tris pH 7.5 (TBS) for 5 minutes.
  • the phage plate from which the filter was taken was stored inverted at 4°C until needed.
  • the TBS buffer was removed from the filter and 5ml of the same buffer plus 3% gelatin with a final pH of 7.5 was added to the filter, and agitated for 15 minutes and drained. Enough TBS buffer containing 3% gelatin, 0.02% azide, and 2 ⁇ g/ml of IgG specific luciferase antibody was added to each filter, which were then gently shaken overnight to permit the antibody to absorb to luciferase antigenic determinants. Next, the filters were washed for 10 minutes with 10ml of TBS buffer, then 5 minutes with 10ml of the same buffer plus 0.05% NP-40, a nonionic detergent. followed by another 10ml of TBS wash.
  • Plaques that adhered to the filters and that exhibit luciferase antigenic determinants were visualized by developing the filters using the chromogenic peroxidase substrate, 4-chloro-l-naphthol. 60ml of the latter were dissolved in 20ml of ice-cold methanol, the latter being mixed just before use with 100ml of TBS containing 60 ⁇ l of
  • the resulting pellet obtained from using 35% - 37% ammonium sulfate was dissolved in a minimal amount of water and dialyzed against the same. The solution was centrifuged and the pellet discarded. Next, the solution was dialyzed against 0.01 molar of sodium phosphate or Tris-Cl buffer, 0.15 molar sodium chloride, pH 7.8 containing 0.02% sodium azide to inhibit bacterial growth.
  • pKJB824.17 exhibits the bacteriophage temperature-sensitive repressor cl 857 . and the Rightward promoter, P r followed by a truncated cro gene exhibiting an Eco RI site.
  • the homology of the nucleotide bases of the 1.8 kilobase fragment produced by Eco RI cleavage of the recombinant phage permits ligation of the fragment into the Eco RI site of pKJB824.17 resulting in fusion of the reading frames of the truncated cro gene to the 1.8 kilobase cDNA fragment.
  • the resulting novel plasmid is termed pKW101 and is shown in Figure 1.
  • pKW101 was transformed into the E. coli strain TBl[ara. ⁇ (lac.proAB),strA. 80lacz ⁇ M15.hsr-,hsm + ] using the calcium precipitation technique described by Maniatis et al., sjipxa.
  • TBI cells containing pKW101 cells were selected by their ability to grow in drug-supplemented media, and then grown up in 10ml of LB broth containing lOg of tryptone, 5g yeast extract, and 5g sodium chloride per liter with pH 7.4. Cells were grown at 30°C to an optical density of 0.8.
  • luciferase activity was achieved by inactivating the ⁇ repressor by heating the cells to 45°C for 30 minutes followed by further incubation for 1 hour at 37°C.
  • Cells were collected by low-speed centrifugation and resuspended in 200 ⁇ l of 100mM KPO 4 pH 8.0, 2mM EDTA, ImM dithiothreitol, 0.2mg/ml protamine sulfate, and lmg/ml lysozyme on ice for 10 minutes. They were frozen on dry ice and thawed to effect cell lysis and hence liberate molecules with luciferase activity.
  • Luciferin alone added to the cell extract yielded a low level of light, presumably because of endogenous levels of ATP in the cells.
  • luciferase activity is not detected in extracts of heat-induced TB1 cells or TB1 cells carrying the vector pKJB824.17 absent cDNA inserts.
  • a suitable plasmid can be constructed by treating the plasmid pKW101 with the restriction endonucleases Sal I and Eco RI so as to remove DNA sequences that encode tetracycline resistance in anticipation of inserting therein a transcription terminator sequence derived from another plasmid.
  • pKW101 minus the region of DNA containing tetracycline-resistance was isolated by agarose electrophoresis. This reaction scheme is shown in Figure 2.
  • the plasmid, pAD9 (also shown in Figure 2), was treated with the restriction endonucleases Bam HI, Eco RI (partial digestion), and then the recessed 3' prime ends created by cleavage with Eco RI and Bam HI filled in with the Klenow fragment of E. coli DNA polymerase 1 as described by Maniatis et al., Supra .
  • the resulting plasmid, termed pKW103 was further treated with Sal I and Eco RI, and the small restriction fragment exhibiting the transcription terminator isolated by agarose gel electrophoresis.
  • plasmid, pKW106 was generated by treating pKW104 with the restriction endonucleases Nru I and Sea I causing the release of a fragment containing cI,P r , the cDNA insert encoding luciferase activity, the transcription termination sequences, and 3' and 5' regions of plasmid DNA.
  • This fragment exhibits the three Hae II sites-one near cl, a second in the DNA region encoding luciferase activity, and the third downstream of the transcription terminator sequence.
  • This fragment was partially digested with Hae II and inserted into the plasmid pUC19, which previously was prepared by partial digestion with Hae II cutting to remove the Lac Z gene contained therein.
  • pKW106 contains in sequential order restriction endonuclease sites for Bam HI, Sma I, and Eco RI immediately downstream of the phage promoter, P r .
  • the DNA region encoding luciferase activity is under control of the phage promoter, P r , and is adjacent the transcription terminator region.
  • the plasmid carries the gene for ampicillin resistance.
  • the restriction sites Bam HI and Sma I are particularly useful in that they are unique and provide sites for inserting cDNA to produce hybrid protein molecules that exhibit luciferase activity plus a second biological activity. Eco RI can also be used for this, but is not unique and requires partial digestion.
  • the plasmid pKW106 was produced as described in Example 5 and treated with Eco RI to release the DNA insert containing the sequence encoding luciferase activity.
  • the sequence was isolated using standard techniques and ligated to the plasmid, pKJB824.11, produced as described by Buckley, suppa after the latter was cut with Eco RI and dephosphorylated with calf intestine alkaline and phosphatase to prevent recirculization and subsequent ligation without a DNA insert.
  • the result is plasmid, pKW108, which is shown in Figure 3.
  • pKW109 Representative of a class of plasmids that express hybrid molecules that exhibit two distinct biological activities is pKW109.
  • This plasmid contains DNA that encodes a hybrid protein exhibiting luciferase activity.
  • Figure 4 shows the steps taken to generate pKW109.
  • the plasmid pKW106 described in Example 5 was restricted with Bam HI and Sea I, and the large fragment containing the DNA sequences, including luciferase activity and the transcription terminator, were isolated.
  • the plasmid pKJB"lac"ZI was cut with Bam HI and Sea I. Restriction with Bam HI and Sea I liberates a fragment with cI,P r and the Lac Z-mc5 DNA sequences. The latter was ligated to the Bam Hl/Sca I DNA fragment produced from pKW106 to yield PKW109.
  • pKW109 in a suitable strain of E. coli can be expected to yield a protein with one enzymatic activity and luciferase, both of which are assayed by techniques described herein, or well-known in the art.
  • pKW109 demonstrates that a large peptide can be fused to Luc without destroying bioluminescent activity. Thus fusions with two enzymatic activities are feasible.
  • hybrid molecules that exhibit luciferase can be employed in are solid-phase immunochemical binding assays to detect antigenic substances in biological fluids.
  • solid-phase immunochemical binding assays to detect antigenic substances in biological fluids.
  • ⁇ -galactosidase using the hybrid molecule, exhibiting luciferase enzymatic activity and ⁇ -galactosidase antigenic activities generated in Example 6.
  • This can be accomplished by raising antibodies to ⁇ -galactosidase; either polyclonal or monoclonal antibodies can be utilized.
  • Polyclonal antibodies can be generated in rabbits and purified by standard ammonium sulfate precipitation procedures, whereas mouse monoclonal antibodies can be prepared by the procedure of Kohler and Milstein as described in Nature. (1975,
  • Antibodies so obtained are affixed to solid support surfaces using techniques well-known in the art, cellulose or agarose beads being suitable for this purpose. Preparation of the beads by cyanogen bromide activation and subsequent ⁇ -galactosidase antibody coupling is carried out as described Wide in Methods of Enzymolooy (1981, 73:203 - 22 Next, beads containing coupled antibody are added to test tubes with a suitable buffer, and a cell extract or other source of fluid containing an unknown amount of ⁇ -galactosidase, and the luciferase ⁇ -galactosidase hybrid molecule added.
  • the reaetants are allowed to compete for binding to antibody bound to the bead for 1 hour at 37°C, then the beads separated from unbound reaetants by centrifugation. After washing the beads with a suitable buffer to remove residual amounts of unbound reaetants, the amount of ⁇ -galactosidase present in the cell extract is determined by assaying the intensity of bioluminescence generated from the luciferase moiety bound indirectly to the beads by attachment of the ⁇ -galactosidase region to the antibody. This is accomplished by performing a luciferase assay as described in Example 1 and monitoring light intensity.
  • Light intensity is related to ⁇ -galactosidase concentration in the cell extract by simultaneously constructing a standard curve by repeating essentially the same assay but using a known amount of ⁇ -galactosidase, and determining the concentrations of ⁇ -galactosidase needed to successfully compete with the hybrid molecule to yield a particular level of light emission.
  • the procedures, buffers, and reaction conditions necessary to carry out these assays is further described by Wide in Methods of Enzymology, supra.
  • DNA from P. pyralis. was isolated from frozen beetles by grinding the same in a mortar and pestle. 50 mM Tris, pH 8, and 10 mM EDTA were added and proteinaceous material digested with proteinase K in the presence of 1% sodium dodecyl sulfate (SDS). DNA was twice extracted, once with phenol-chloroform and a second time with chloroform, and precipitated with ethanol and redissolved in TBS buffer. The DNA was subsequently purified by banding on an ethidium bromide-cesium chloride equilibrium gradient. After isolating the same from the gradient, DNA was subjected to partial digestion with the restriction endonuclease Sau3A.
  • Fragments ranging in size from 12 - 20 kilobases were isolated by centrifugation on a sucrose-density gradient. These fragments were ligated to the ⁇ vector EMBL4 DNA, which was previously digested with Bam HI. This phage mixture was packaged using ⁇ in vitro packaging extracts, which are commercially available form Bethesda Research Laboratories, or Vector Cloning Systems. Packaged phage were then plated on a lawn of E. coli cells, strained LE392, and plaques so obtained were screened by the filter hybridization method of Benton and Davis as described in Science (1977, 191:180 - 182).
  • the probe used to detect plaques harboring luciferase DNA sequences was a 5' Eco RI fragment obtained from the cDNA library discussed in Example 4. This probe, termed Luc23, was labeled with 32 P by nick- translation as described by Rigby, Diekman, Rose, and Berg in Journal of Molecular Biology (1977, 113: 237 - 251). Plaques were detected that hybridized to the Luc23 cDNA probe, and these were purified by standard techniques. All the clones obtained contained three Eco RI fragments that were homologous to the luciferase Luc23 cDNA probe. A restriction map of the luciferase gene is shown in Figure 5.
  • a restriction map of pJD200 is shown in Figure 6. Digestion of pJD200 with XbAI releases all but the 5'-most sequences of the luciferase genomic DNA from the vector. Thus, when pJD200 was digested with Bam HI and XbAI, it was possible to isolate the fragment consisting of the vector plus the 5'-most portion of the luciferase gene. This fragment was ligated to a Bam HI-XbAI fragment composed of luciferase cDNA extending from the same Xbal site that was present in the genomic DNA to the end 3' of the Luc23 cDNA to yield the plasmid pJD201.
  • pJD201 Unlike the Luc23 cDNA, which lacks an initiation codon, pJD201 has the natural luciferase translational start and polyadenylation site. Plasmid pJD201 is shown in Figure 7. It should be noted that the Luc23 cDNA/genomic insert of pJD201 is suitable in use with a wide variety of vectors that may be expressed in eucaryotes and procaryotes. Further, it should be noted that the hybrid DNA molecule can also be utilized for expressing luciferase activity in plant cells.
  • the Hind III-Bam HI DNA fragment exhibiting luciferase sequences was obtained from pJD201 described in Example 9 using the same enzymes, and was inserted downstream from an SV 40 promoter in the plasmid pBR322-based vector pSV2.
  • the new construct, termed plasmid pSV2 L was introduced into CV 1 monkey cells by the calcium phosphate DNA precipitation technique. After 48 hours, cells were harvested and lysed by three cycles of freezing and thawing in 0.1 molar potassium phosphate (KPO 4 ), PH 7.8, containing ImM dithiothreitol. The cell extracts were assayed for luciferase activity as described in Example 4. Western blot analysis revealed that the hybrid DNA construct, Luc23 cDNA- genomic DNA, synthesizes native luciferase.

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US5422266A (en) * 1984-12-31 1995-06-06 University Of Georgia Research Foundation, Inc. Recombinant DNA vectors capable of expressing apoaequorin
US4968613A (en) * 1987-07-29 1990-11-06 Kikkoman Corporation Luciferase gene and novel recombinant DNA as well as a method of producing luciferase
US5182202A (en) * 1987-11-30 1993-01-26 Kikkoman Corporation Purified luciferase from luciola cruciata
JPH088863B2 (ja) * 1987-11-30 1996-01-31 キッコーマン株式会社 ルシフェラーゼ
AU3342689A (en) * 1988-03-24 1989-10-16 Igen Incorporated Luminescent chimeric proteins
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DE68910036T2 (de) * 1988-07-01 1994-03-31 Kikkoman Corp Luciferase-Gen und neue rekombinante DNA sowie Verfahren zur Herstellung von Luciferase.
JPH0771485B2 (ja) * 1988-09-01 1995-08-02 キッコーマン株式会社 ルシフェラーゼの製造法
DE68922971T2 (de) * 1988-12-06 1995-10-19 Chisso Corp Mit einem Protein mit spezifischer Bindungsaktivität fusioniertes Aequorin, seine Herstellung, Reinigung und Nachweismethode.
US5292658A (en) * 1989-12-29 1994-03-08 University Of Georgia Research Foundation, Inc. Boyd Graduate Studies Research Center Cloning and expressions of Renilla luciferase
ES2142801T3 (es) * 1991-03-11 2000-05-01 Univ Georgia Res Found Clonacion y expresion de luciferasa de renilla.
AU5734298A (en) * 1997-02-06 1998-08-26 Aventis Pharmaceuticals Inc. Human nerve growth factor exon 1 and exon 3 promoters
US6673554B1 (en) * 1999-06-14 2004-01-06 Trellie Bioinformatics, Inc. Protein localization assays for toxicity and antidotes thereto
US7109315B2 (en) 2000-03-15 2006-09-19 Bruce J. Bryan Renilla reniformis fluorescent proteins, nucleic acids encoding the fluorescent proteins and the use thereof in diagnostics, high throughput screening and novelty items
US6855510B2 (en) * 2001-03-20 2005-02-15 Dana Farber Cancer Institute, Inc. Pharmaceuticals and methods for treating hypoxia and screening methods therefor
US7919274B2 (en) * 2001-03-20 2011-04-05 Dana-Farber Cancer Institute, Inc. Light-emitting fusion proteins and diagnostic and therapeutic methods therefor

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