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WO2000034318A1 - Proteines fluorescentes d'especes non bioluminescentes de la classe des anthozoaires, genes codant pour ces proteines et utilisations de ces proteines - Google Patents

Proteines fluorescentes d'especes non bioluminescentes de la classe des anthozoaires, genes codant pour ces proteines et utilisations de ces proteines Download PDF

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WO2000034318A1
WO2000034318A1 PCT/US1999/029291 US9929291W WO0034318A1 WO 2000034318 A1 WO2000034318 A1 WO 2000034318A1 US 9929291 W US9929291 W US 9929291W WO 0034318 A1 WO0034318 A1 WO 0034318A1
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dna
isolated
fluorescent protein
encodes
organism
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WO2000034318A9 (fr
Inventor
Sergey Anatolievich Lukyanoy
Arcady Fedorovich Fradkov
Yulii Aleksandrovich Labas
Mikhail Vladimirovich Matz
Xin Jiang
Tommy Duong
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Takara Bio USA Inc
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Clontech Laboratories Inc
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/43504—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/43504—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates
    • C07K14/43595—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from coelenteratae, e.g. medusae
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/05—Animals comprising random inserted nucleic acids (transgenic)

Definitions

  • This invention relates to the field of molecular biology. More specifically, this invention relates to novel fluorescent proteins , cDNAs encoding the proteins and uses thereof.
  • Fluorescence labeling is a particularly useful tool for marking a protein, cell, or organism of interest.
  • a protein of interest is purified, then covalently conjugated to a fluorophore derivative.
  • the protein-dye complex is then inserted into cells of interest using micropipetting or a method of reversible permeabilization.
  • the dye attachment and insertion steps make the process laborious and difficult to control.
  • An alternative method of labeling proteins of interest is to concatenate o r fuse the gene expressing the protein of interest to a gene expressing a marker, then express the fusion product.
  • Typical markers for this method of protein labeling include ⁇ -galactosidase, firefly luciferase and bacterial luciferase. These markers, however, require exogenous substrates or cofactors and are therefore of limited use for in vivo studies .
  • a marker that does not require an exogenous cofactor o r substrate is the green fluorescent protein (GFP) of the jellyfish Aequorea victoria, a protein with an excitation maximum at 395 nm, a second excitation peak at 475 nm and an emission maximum at 5 1 0 nm.
  • GFP is a 238-amino acid protein, with amino acids 65-67 involved in the formation of the chromophore.
  • GFP GFP-binding protein
  • GFP expression in plant cells is discussed by Hu and Cheng in Febs Letters 369 ( 1995 ) , 331-334, while GFP expression in Drosophila embryos is described by Davis et al. in Dev . Biology 170 (1995), 726-729. Crystallographic structures of wild-type GFP and the mutant
  • GFP S65T reveal that the GFP tertiary structure resembles a barrel (Ormo et al., Science 273 (1996), 1392- 1395; Yang, et al., Nature Biotechnol 14 ( 1996), 1246- 1251 ).
  • the barrel consists of beta sheets in a compact structure, where, in the center, an alpha helix containing the chromophore is shielded by the barrel.
  • the compact structure makes GFP very stable under diverse and/or harsh conditions such as protease treatment, making GFP an extremely useful reporter in general. However, the stability of GFP makes it sub-optimal for determining short-term or repetitive events.
  • GFP GFP reagents useful and optimized for a variety of research purposes.
  • New versions of GFP have been developed, such as a "humanized” GFP DNA, the protein product of which has increased synthesis in mammalian cells (Haas, et al., Current Biology 6 (1996), 315-324; Yang, et al., Nucleic Acids Research 24 ( 1996), 4592-4593).
  • EGFP enhanced green fluorescent protein
  • Other mutations to GFP have resulted in blue-, cyan- and yellow-green light emitting versions.
  • Novel fluorescent proteins result in possible new colors, or produce pH-dependent fluorescence.
  • Other benefits of novel fluorescent proteins include fluorescence resonance energy transfer (FRET) possibilities based on new spectra and better suitability for larger excitation.
  • FRET fluorescence resonance energy transfer
  • the prior art is deficient in novel fluorescent proteins wherein the DNA coding sequences are known.
  • the present invention fulfills this long-standing need in the art.
  • the present invention is directed to DNA sequences encoding fluorescent proteins selected from the group consisting of: (a) an isolated DNA from an organism from the Class Anthozoa which encodes a fluorescent protein; (b) an isolated DNA which hybridizes t o the isolated DNA of (a) and which encodes a fluorescent protein; and (c) an isolated DNA differing from the isolated DNAs of (a) and (b) in codon sequence due to the degeneracy of the genetic code and that encodes a fluorescent protein.
  • the DNA is isolated from a non -bioluminescent organism from Class Anthozoa. More preferably, the DNA has the sequence selected from the group consisting of SEQ ID Nos. 55, 57-60, and the fluorescent protein has the amino acid sequence shown in SEQ ID No. 56.
  • a vector capable of expressing the DNA of the present invention in a recombinant cell comprising said DNA and regulatory elements necessary for expression of the DNA in the cell.
  • the DNA encodes a fluorescent protein having the amino acid sequence shown in SEQ ID No. 56.
  • a host cell transfected with a vector of the present invention, such that the host cell expresses a fluorescent protein .
  • the cell is selected from the group consisting of bacterial cells, mammalian cells, plant cells and insect cells.
  • the present invention is also directed to an isolated and purified fluorescent protein coded for by DNA selected from the group consisting of: (a) isolated DNA from an organism from Class Anthozoa which encodes a fluorescent protein; (b) isolated DNA which hybridizes to the isolated DNA of (a) and which encodes a fluorescent protein; and (c) isolated DNA differing from the isolated DNAs of (a) and (b) in codon sequence due to the degeneracy of the genetic code, and which encodes a fluorescent protein.
  • the protein has the amino acid sequence shown in SEQ ID No. 56.
  • the present invention is also directed to a DNA sequence encoding a fluorescent protein selected from the group consisting of: (a) an isolated DNA which encodes a fluorescent protein, wherein said DNA is from an organism from Class Anthozoa and wherein said organism does not exhibit bioluminescence; (b) an isolated DNA which hybridizes to isolated DNA of (a) and which encodes a fluorescent protein; and (c) an isolated DNA differing from the isolated DNAs of (a) and (b) in codon sequence due to degeneracy of the genetic code and which encodes a fluorescent protein.
  • the organism is from Sub-class Zoantharia, Order Zoanthidea. More preferably, th e organism is from Sub-order Brachycnemina. Even more preferably, the organism is from Family Zoanthidae, Genus Zoanthus.
  • the present invention is drawn to a novel fluorescent protein from Zoanthus sp. , zFP506.
  • the present invention is further directed to an amino acid sequence which can be used as a basis for designing an oligonucleotide probe for identification of a DNA encoding a fluorescent protein b y means of hybridizaton, wherein the amino acid sequence is selected from the group consisting of SEQ ID Nos. 3, 5, 8, 11, 12, 14.
  • amino acid sequence is selected from the group consisting of SEQ ID Nos. 3, 5, 8, 11, 12, 14.
  • such an oligonucleotide has a nucleotide sequence selected from th e group consisting of SEQ ID Nos. 4, 6, 7, 9, 10, 13, 15, 16.
  • Figure 1 shows the modified strategy of 3'-RACE used t o isolate the target fragments. Sequences of the oligonucleotides used are shown in Table 2. Dpi and Dp2 are the degenerate primers used in the first and second PCR, respectively (see Tables 3 and 4 for the sequences of degenerate primers). In the case of Zoanthus sp., the first degenerate primer used was NGH (SEQ ID No. 4), and the second degenerate primer used was GEGa (SEQ ID No. 6). Figure 2 shows the excitation and emission spectrum of the novel fluorescent protein from Zoanthus sp. , zFP506.
  • Figure 3 shows functional analysis of destabilized zFP506. It demonstrates that fusion of MODC domain to the zFP506 slightly decreases the fluorescent intensity compared to zFP506 itself and that transient transfection of destabilized zFP506 decreases the fluorescent intensity by 50% after 4-hour treatment with protein synthesis inhibitor cycloheximide ( Figure 3B) vs. control ( Figure 3A) .
  • GFP refers to the basic green fluorescent protein from Aequorea victoria, including prior art versions of GFP engineered to provide greater fluorescence or fluoresce in different colors.
  • sequence of Aequorea victoria GFP SEQ ID No.
  • EGFP refers to mutant variant o f GFP having two amino acid substitutions: F64L and S65T (Heim et al., Nature 373 (1995), 663-664).
  • humanized refers to changes made to the GFP nucleic acid sequence to optimize the codons for expression of the protein in human cells (Yang et al., Nucleic Acids Research 24 ( 1996), 4592-4593).
  • NFP novel fluorescent protein
  • GNFP green novel fluorescent protein
  • GNFP green novel fluorescent protein
  • GNFP zFP506.
  • a "vector” is a replicon, such as plasmid, phage or cosmid, to which another DNA segment may be attached so as to bring about the replication of the attached segment.
  • a “DNA molecule” refers to the polymeric form of deoxyribonucleotides (adenine, guanine, thymine, or cytosine) in either single stranded form or a double-stranded helix. This term refers only to the primary and secondary structure of the molecule, and does no t limit it to any particular tertiary forms. Thus, this term includes double-stranded DNA found, inter alia, in linear DNA molecules (e.g. , restriction fragments), viruses, plasmids, and chromosomes.
  • a DNA "coding sequence” is a DNA sequence which is transcribed and translated into a polypeptide in vivo when placed under the control of appropriate regulatory sequences.
  • a coding sequence can include, but is not limited to, prokaryotic sequences, cDNA from eukaryotic mRNA, genomic DNA sequences from eukaryotic (e.g., mammalian) DNA, and synthetic DNA sequences.
  • a polyadenylation signal and transcription termination sequence may be located 3' to the coding sequence.
  • hybridization refers to the process of association of two nucleic acid strands to form a n antiparallel duplex stabilized by means of hydrogen bonding between • residues of the opposite nucleic acid strands.
  • oligonucleotide refers to a short (under 1 00 bases in length) nucleic acid molecule.
  • DNA regulatory sequences are transcriptional and translational control sequences, such as promoters , enhancers, polyadenylation signals, terminators, and the like, that provide for and/or regulate expression of a coding sequence in a ho s t cell.
  • a “promoter sequence” is a DNA regulatory region capable of binding RNA polymerase in a cell and initiating transcription of a downstream (3' direction) coding sequence.
  • the promoter sequence is bounded at its 3 ' terminus by the transcription initiation site and extends upstream ( 5 ' direction) to include the minimum number of bases or elements necessary to initiate transcription at levels detectable above background.
  • a transcription initiation site within the promoter sequence will be found a transcription initiation site, as well as protein binding domains responsible for the binding of RNA polymerase.
  • Eukaryotic promoters will often, but not always, contain "TATA" boxes and "CAT” boxes .
  • Various promoters, including inducible promoters may be used t o drive the various vectors of the present invention.
  • restriction endonucleases and “restriction enzymes” refer to bacterial enzymes, each of which cut double-stranded DNA at or near a specific nucleotide sequence.
  • a cell has been "transformed” or “transfected” by exogenous or heterologous DNA when such DNA has been introduced inside the cell.
  • the transforming DNA may or may not be integrated (covalently linked) into the genome of the cell.
  • the transforming DNA may b e maintained on an episomal element such as a plasmid.
  • a stably transformed cell is one in which the transforming DNA has become integrated into a chromosome so that it is inherited by daughter cells through chromosome replication.
  • a "clone” is a population o f cells derived from a single cell or common ancestor by mitosis.
  • a "cell line” is a clone of a primary cell that is capable of stable growth in vitro for many generations.
  • heterologous region of the DNA construct is a n identifiable segment of DNA within a larger DNA molecule that is no t found in association with the larger molecule in nature.
  • the heterologous region encodes a mammalian gene
  • the gene will usually be flanked by DNA that does not flank the mammalian genomic DNA in the genome of the source organism.
  • heterologous DNA includes coding sequence in a construct where portions of genes from two different sources have been brought together so as to produce a fusion protein product. Allelic variations or naturally-occurring mutational events do not give rise to a heterologous region of DNA as defined herein.
  • reporter gene refers to a coding sequence attached to heterologous promoter or enhancer elements and whose product may be assayed easily and quantifiably when the construct is introduced into tissues or cells.
  • the amino acids described herein are preferred to be in the
  • L isomeric form.
  • the amino acid sequences are given in one-letter code (A: alanine; C: cysteine; D: aspartic acid; E: gluetamic acid; F: phenylalanine; G: glycine; H: histidine; I: isoleucine; K: lysine; L: leucine; M: metionine; N: asparagine; P: proline; Q: gluetamine; R: arginine; S: serine; T: threonine; V: valine; W: tryptophane; Y: tyrosine; X: any residue).
  • NH2 refers to the free amino group present at the amino terminus of a polypeptide.
  • COOH refers to the free carboxy group present at the carboxy terminus of a polypeptide. In keeping with standard polypeptide nomenclature, / Biol. Chem ., 243 ( 1969), 3552- 59 is used.
  • the present invention is directed to an isolated DNA selected from the group consisting of: (a) isolated DNA from a n organism from the Class Anthozoa which encodes a fluorescent protein; (b) isolated DNA which hybridizes to isolated DNA of (a) and which encodes a fluorescent protein; and (c) isolated DNA differing from the isolated DNAs of (a) and (b) in codon sequence due to th e degeneracy of the genetic code, and which encodes a fluorescent protein.
  • the DNA has the sequence selected from the group consisting of SEQ ID Nos. 55, 57-60, and the fluorescent protein has th e amino acid sequence shown in SEQ ID No. 56. More preferably, th e DNA is non-humanized or humanized zFP506 or N65M.
  • a vector capable of expressing the DNA of the present invention in a recombinant cell comprising said DNA and regulatory elements necessary for expression of the DNA in the cell.
  • the DNA encodes a fluorescent protein having the amino acid sequence shown in SEQ ID No. 56.
  • the vector is constructed by amplifying the DNA and then inserting the amplified DNA into EGFP-Nl backbone, or by fusing different mouse ODC degradation domains such as dl , d2 and d376 to the C-terminal of the DNA and then inserting th e fusion DNA into EGFP-Nl backbone.
  • a host cell transfected with the vector of the present invention which expresses a fluorescent protein of the present invention.
  • the cell is selected from the group consisting of bacterial cells, mammalian cells, plant cells, insect cells and yeast cells.
  • a representative example of mammalian cell is HEK 293 cell and a n example of bacterial cell is an E. coli cell.
  • the present invention is also directed to a DNA sequence encoding a fluorescent protein selected from the group consisting of: (a) an isolated DNA which encodes a fluorescent protein, wherein said DNA is from an organism from Class Anthozoa and wherein said organism does not exhibit bioluminescence; (b) an isolated DNA which hybridizes to isolated DNA of (a) and which encodes a fluorescent protein; and (c) an isolated DNA differing from the isolated DNAs of (a) and (b) in codon sequence due to degeneracy of the genetic code and which encodes a fluorescent protein.
  • the organism is from Sub-class Zoantharia, Order Zoanthidea. More preferably, th e organism is from Sub-order Brachycnemina. Even more preferably, the organism is from Family Zoanthidae, Genus Zoanthus.
  • the present invention is also directed to an isolated an d purified fluorescent protein coded for by DNA selected from the group consisting of: (a) an isolated protein encoded by a DNA which encodes a fluorescent protein wherein said DNA is from an organism from Class Anthozoa and wherein said organism does not exhibit bioluminescence; (b) an isolated protein encoded by a DNA which hybridizes to isolated DNA of (a); and (c) an isolated protein encoded by a DNA differing from the isolated DNAs of (a) and (b) in codon sequence due t o degeneracy of the genetic code.
  • the isolated and purified fluorescent protein is zFP506.
  • the present invention is further directed to an amino acid sequence which can be used as a basis for designing an oligonucleotide probe for identification of a DNA encoding a fluorescent protein by means of hybridizaton, wherein the amino acid sequence is selected from the group consisting of SEQ ID Nos. 3, 5, 8, 11, 12, 14.
  • the amino acid sequence is selected from the group consisting of SEQ ID Nos. 3, 5, 8, 11, 12, 14.
  • such an oligonucleotide has a nucleotide sequence selected from th e group consisting of SEQ ID Nos. 4, 6, 7, 9, 10, 13, 15, 16 and is used as a primer in polymerase chain reaction.
  • it can be used a s a probe for hybridization screening of the cloned genomic or cDNA library.
  • Novel fluorescent proteins were identified from several genera of Anthozoa which do not exhibit any bioluminescence but have fluorescent color as observed under usual white light or ultraviolet light. Six species were chosen (see Table 1).
  • Amplified cDNA samples were then prepared as described in the protocol provided except the two primers used for PCR were the TS primer (5'-AAGCAGTGGTATCAACGCAGAGT, SEQ ID No. 2 ) (Table 2) and the TN3 primer (Table 2), both in 0J ⁇ M concentration. Twenty to twenty-five PCR cycles were performed to amplify a cDNA sample. The amplified cDNA was diluted 20-fold in water and 1 ⁇ l of this dilution was used in subsequent procedures.
  • T7-TN3 5 '-GTAATACGACTC ACT ATAGGGCCGC AGTCG ACCG(T) 13
  • T7-TS 5'-GTAATACGACTCACTATAGGGCAAGCAGTGGTATCAACGCAGAGT
  • PCR using degenerate primers was performed.
  • Degenerate primers were designed to match the sequence of the mRNAs in regions that were predicted to be the most invariant in the family of fluorescent proteins. Four such stretches were chosen (Table 3) and variants of degenerate primers were designed. All such primers were directed t o the 3 '-end of mRNA. All oligos were gel-purified before use. Table 2 shows the oligos used in cDNA synthesis and RACE.
  • the modified strategy of 3 '-RACE was used to isolate the target fragments (see Figure 1).
  • the RACE strategy involved two consecutive PCR steps.
  • the first PCR step involved a first degenerate primer (Table 4) and the T7-TN3 primer (SEQ ID No. 17) which has a 3 ' portion identical to the TN3 primer used for cDNA synthesis (for sequence of T7-TN3, Table 2).
  • the reason for substituting the longer T7-TN3 primer in this PCR step was that background amplification which occurred when using the shorter TN3 primer was suppressed effectively, particularly when the T7-TN3 primer was used at a low concentration (0.1 _M) (Frohman et al., (1998) /WAS USA, 85, 8998- 9002).
  • the second PCR step involved the TN3 primer (SEQ ID No. 1 , Table 2) and a second degenerate primer (Table 4).
  • NGH GEGa SEQ ID No. 6
  • SEQ ID No. 4 NFP (SEQ ID No. 13) or PVMb (SEQ ID No. 16)
  • the first PCR reaction was performed as follows: 1 ⁇ l of 20-fold dilution of the amplified cDNA sample was added into the reaction mixture containing IX Advantage KlenTaq Polymerase Mix with provided buffer (CLONTECH), 200 ⁇ M dNTPs, 0.3 ⁇ M of first degenerate primer (Table 4) and 0.1 ⁇ M of T7-TN3 (SEQ ID No. 17) primer in a total volume of 20 ⁇ l.
  • the cycling profile was (Hybaid OmniGene Thermocycler, tube control mode): 1 cycle for 95°C, 10 sec; 55°C, 1 min.; 72°C, 40 sec; 24 cycles for 95°C, 10 sec; 62°C, 30 sec; 72°C, 40 sec.
  • reaction was then diluted 20-fold in water and 1 ⁇ l of this dilution was added to a second PCR reaction, which contained IX Advantage KlenTaq Polymerase Mix with the buffer provided by the manufacturer (CLONTECH), 200 ⁇ M dNTPs, 0.3 ⁇ M of the second degenerate primer (Table 4) and 0J ⁇ M of TN3 primer.
  • the cycling profile was (Hybaid OmniGene Thermocycler, tube control mode): 1 cycle for 95°C, 10 se ; 55°C (for GEG/GNG or PVM) or 52°C (for NFP), 1 min.; 72°C, 40 sec; 13 cycles for 95°C, lOsec; 62°C (for GEG/GNG o r PVM) or 58°C (for NFP), 30 sec; 72°C, 40 sec.
  • the product of PCR was cloned into PCR-Script vector (Stratagene) according to the manufacturer' s protocol.
  • the step-out reaction mixture contained lx Advantage KlenTaq Polymerase Mix using buffer provided by the manufacturer (CLONTECH), 200 ⁇ M dNTPs, 0.2 ⁇ M of the first gene-specific primer (see Table 5), 0.02 ⁇ M of the T7-TS primer (SEQ ID No. 18), 0.1 ⁇ M of T7 primer (SEQ ID No.
  • the cycling profile was (Hybaid OmniGene Thermocycler, tube control mode): 23 -27 cycles for 95°C, 10 sec; 60°C, 30 sec; 72°C, 40 sec.
  • the product of amplification was diluted 50-fold in water and one ⁇ l of this dilution was added to the second (nested) PCR.
  • the reaction contained IX Advantage KlenTaq Polymerase Mix with provided buffer (CLONTECH), 200 ⁇ M dNTPs, 0.2 ⁇ M of the second gene-specific primer and 0J ⁇ M of TS primer (SEQ ID No.
  • Both primers had 5 '-heels coding for a site for a restriction endonuclease; in addition, the upstream primer was designed so as to allow the cloning of the PCR product into the pQE30 vector (Qiagen) in such a way that resulted in the fusion of reading frames of the vector-encoded 6xHis-tag and nFP.
  • the PCR was performed as follows: 1 ⁇ l of the 20-fold dilution of the amplified cDNA sample was added to a mixture containing lx Advantage KlenTaq Polymerase Mix with buffer provided by the manufacturer (CLONTECH), 200 ⁇ M dNTPs, 0.2 ⁇ M of upstream primer and 0.2 ⁇ M of downstream primer, in a final total volume of 20 ⁇ l.
  • the cycling profile was (Hybaid OmniGene Thermocycler, tube control mode): 23-27 cycles for 95°C , 10 sec; 60°C, 30 sec; 72°C, 40 sec.
  • the product of this amplification step was purified by phenol-chlorophorm extraction and ethanol precipitation and then cloned into pQE30 vector using restriction endonucleases corresponding to the primers' sequence according t o standard protocols.
  • All plasmids were amplified in XL-1 blue E. coli and purified by plasmid DNA miniprep kits (CLONTECH).
  • the recombinant clones were selected by colony color, and grown in 3 ml of LB medium (supplemented with 100 ⁇ g/ml of ampicillin) at 37°C overnight. 100 ⁇ l of the overnight culture was transferred into 200 ml of fresh IB medium containing 100 ⁇ g/ml of ampicillin and grown at 37°C, 200 m up to OD 600 0.6-0.7. 1 mM IPTG was then added to the culture and incubation was allowed to proceed at 37°C for another 16 hours.
  • the cells were harvested and recombinant protein, which incorporated 6x His tags on the N-terminus, was purified using TALONTM metal-affinity resin according to the manufacturer's protocol (CLONTECH).
  • zFP506 One of the full-length cDNAs encoding fluorescent proteins found is described herein (zFP506).
  • the nucleic acid sequence and deduced amino acid sequence are SEQ ID Nos. 55 and 56, respectively.
  • the spectral properties of zFP506 is listed in Table 7, and the emission and excitation spectra for the ZFP506 is shown in Figure 2.
  • *relative brightness is extinction coefficient multiplied by quantum yield divided by the same value for A. victoria GFP .
  • N65M One mutant of zFP506 was generated, N65M. Compared with wild type zFP506, N65M has the mutation of from "AAC” to "ATG” which results in the corresponding amino acid change from Asparagine (N) to Methionine (M) at the position of 65.
  • the spectral properties of N65M are listed in Table 8.
  • *relative brightness is extinction coefficient multiplied by quantum yield divided by the same value for A. victoria GFP.
  • Non-humanized zFP506 DNA was amplified via PCR and reconstructed into EGFP-Nl backbone. This vector has the s ame multiple cloning sites as EGFP-Nl .
  • Destabilized 7.FP506 Vectors as Transcription Reporters Since zFP506 is very stable, it is necessary to generate destabilized versions of zFP506 in order to observe the rapid turnover of the protein.
  • two destabilized zFP506 vectors were constructed by fusing mouse ODC degredation domain to the C-terminal of zFP506.
  • the dl version of destablized zFP506 (SEQ ID No. 57) has three E to A mutations within MODC degredation domain comparing to d2 version (SEQ ID No. 58 ) , therefore result in a shorter half-life of the protein to which MODC degradation domain fused to.
  • Destablized dlzFP506 and d2zFP506 were constructed in EGFP-Nl backbone
  • Destabilized dlzFP506 was constructed into pCRE-dlGNFP and pNF- ⁇ B-dlGNFP vectors. Its expression was regulated under cAMP response element (CRE) or NF- ⁇ B response element, respectively. These vectors were transiently transfected into 293 cells, and 24 hours post transfection, the expression of dlGNFP was induced by Forskolin o r TNF- ⁇ . 6 hours after induction, the culture was analysed by FACS. CRE- dlGNFP showed 7 fold of induction in fluorescence intensity, while 4 fold of induction was obtained in NF- ⁇ B-dlGNFP (data not shown). This demonstrated that the destabilized form of GNFP is applicable as transcription reporters.
  • CRE cAMP response element
  • NF- ⁇ B response element NF- ⁇ B response element
  • Humanized N65M Since mammalian expression is a very popular tool, human favored codon version is needed for better expression in mammalian cells. Each piece of human favored codon oligos was linked to form th e full length of wild type and/or mutant zFP506 (hGNFP-zFP506, SEQ ID No. 59; hGNFP-N65M, SEQ ID No. 60). This humanized zFP506 was constituted into EGFP-Nl backbone.

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Abstract

L'invention concerne de nouvelles protéines fluorescentes d'organismes non bioluminescents de la classe des anthozoaires. L'invention concerne également des ADNc codant pour ces protéines fluorescentes.
PCT/US1999/029291 1998-12-11 1999-12-10 Proteines fluorescentes d'especes non bioluminescentes de la classe des anthozoaires, genes codant pour ces proteines et utilisations de ces proteines Ceased WO2000034318A1 (fr)

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WO2002059309A3 (fr) * 2000-12-13 2003-03-13 Clontech Lab Inc Chromoproteines derivees d'anthozoaire, mutants fluorescents de celles-ci et procedes d'utilisation associes
JP2003527833A (ja) * 1999-10-14 2003-09-24 クロンテック・ラボラトリーズ・インコーポレーテッド 花虫類に由来する発色団／蛍光体、およびそれらの使用法
EP1135532A4 (fr) * 1998-12-11 2004-11-03 Clontech Lab Inc Proteines fluorescentes appartenant a des especes non bioluminescentes de la classe des anthozoaires, genes codant pour ces proteines et utilisations
WO2004111235A1 (fr) * 2003-06-16 2004-12-23 Riken Proteine fluorescente et proteine pigment
US6969597B2 (en) 2001-02-21 2005-11-29 Clontech Laboratories, Inc. Nucleic acids encoding non aggregating fluorescent proteins and methods for using the same
US6977293B1 (en) 2000-11-03 2005-12-20 Ceres, Inc. Chimeric polypeptides
EP1372418A4 (fr) * 2001-02-26 2006-01-25 Univ Proteines fluorescentes tandem non oligomerisantes
US7005511B2 (en) 2001-02-26 2006-02-28 The Regents Of The University Of California Fluorescent protein variants and methods for making same
US7157565B2 (en) 2000-10-12 2007-01-02 Clontech Laboratories, Inc. Far red shifted fluorescent proteins
US7183399B2 (en) 2001-10-12 2007-02-27 Clontech Laboratories, Inc. Nucleic acids encoding linked chromo/fluorescent domains and methods for using the same
WO2007059077A2 (fr) 2005-11-14 2007-05-24 E.I.Du Pont De Nemours And Company Compositions et methodes d'alteration du contenu alpha- et beta-tocotrienol
US7338785B2 (en) 1998-12-11 2008-03-04 Clontech Laboratories, Inc. Nucleic acids encoding chromophores/fluorophores and methods for using the same
US7344886B2 (en) 2002-11-29 2008-03-18 Boehringer Ingelheim Pharma Gmbh & Co., Kg Neomycin-phosphotransferase-genes and methods for the selection of recombinant cells producing high levels of a desired gene product
EP1918364A2 (fr) 2003-08-19 2008-05-07 Boehringer Ingelheim Pharma GmbH & Co. KG Procedé de reclonage de cellules de production
US7384744B2 (en) 2002-11-29 2008-06-10 Boehringer Ingelheim Pharma Gmbh & Co., Kg Expression vector, methods for the production of heterologous gene products and for the selection of recombinant cells producing high levels of such products
US7393923B2 (en) 2004-11-08 2008-07-01 The Regents Of The University Of California Red-shifted fluorescent proteins mPlum and mRaspberry and polynucleotides encoding the same
US7479555B2 (en) 1999-07-21 2009-01-20 Ceres, Inc. Polynucleotides having a nucleotide sequence that encodes a polypeptide having MOV34 family activity
US7485715B2 (en) 1999-06-18 2009-02-03 Ceres, Inc. Sequence-determined DNA encoding AP2 domain polypeptides
US7537915B2 (en) 1998-12-11 2009-05-26 Clontech Laboratories, Inc. Nucleic acids encoding chromophores/fluorophores and methods for using the same
US7678893B2 (en) 2002-12-26 2010-03-16 Zakrytoe Aktsionernoe Obschestvo “Evrogen” Fluorescent proteins from Copepoda species and methods for using same
US7687614B2 (en) 2001-02-26 2010-03-30 The Regents Of The University Of California Monomeric and dimeric fluorescent protein variants and methods for making same
DE102010018878A1 (de) 2009-04-30 2010-11-04 Julius-Maximilians-Universität Würzburg Neue Zell-Linie zur Fluoreszenz-basierten Detektion von funktionell aktiven Antikörpern und Autoantikörpern gegen den Beta1-adrenergen Rezeptor
EP2284195A1 (fr) 2001-09-18 2011-02-16 Carnegie Institution Of Washington Protéines hybrides destinées à la détection d'analytes
US8084575B2 (en) 2002-12-20 2011-12-27 Julius-Maximilians-Universitat Millisecond activation switch for seven-transmembrane proteins
US8669109B2 (en) 2003-08-19 2014-03-11 Boehringer Ingelheim Pharma Gmbh & Co. Kg Methods of producing proteins in Chinese hamster ovary (CHO) cells
US9045776B2 (en) 2006-07-26 2015-06-02 Boehringer Ingelheim Pharma Gmbh & Co. Kg Regulatory nucleic acid elements
US9758790B2 (en) 2004-12-08 2017-09-12 Ceres, Inc. Modulating the level of components within plants
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US9834584B2 (en) 1998-12-11 2017-12-05 Takara Bio Usa, Inc. Nucleic acids encoding chromophores/fluorophores and methods for using the same
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US7485715B2 (en) 1999-06-18 2009-02-03 Ceres, Inc. Sequence-determined DNA encoding AP2 domain polypeptides
US7479555B2 (en) 1999-07-21 2009-01-20 Ceres, Inc. Polynucleotides having a nucleotide sequence that encodes a polypeptide having MOV34 family activity
US7166444B2 (en) 1999-10-14 2007-01-23 Clontech Laboratories, Inc. Nucleic acids encoding chromophores/fluorophores and methods for using the same
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JP2003527833A (ja) * 1999-10-14 2003-09-24 クロンテック・ラボラトリーズ・インコーポレーテッド 花虫類に由来する発色団／蛍光体、およびそれらの使用法
US7157565B2 (en) 2000-10-12 2007-01-02 Clontech Laboratories, Inc. Far red shifted fluorescent proteins
US6977293B1 (en) 2000-11-03 2005-12-20 Ceres, Inc. Chimeric polypeptides
WO2002059309A3 (fr) * 2000-12-13 2003-03-13 Clontech Lab Inc Chromoproteines derivees d'anthozoaire, mutants fluorescents de celles-ci et procedes d'utilisation associes
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US8084575B2 (en) 2002-12-20 2011-12-27 Julius-Maximilians-Universitat Millisecond activation switch for seven-transmembrane proteins
US7678893B2 (en) 2002-12-26 2010-03-16 Zakrytoe Aktsionernoe Obschestvo “Evrogen” Fluorescent proteins from Copepoda species and methods for using same
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EP1918364A2 (fr) 2003-08-19 2008-05-07 Boehringer Ingelheim Pharma GmbH & Co. KG Procedé de reclonage de cellules de production
US8669109B2 (en) 2003-08-19 2014-03-11 Boehringer Ingelheim Pharma Gmbh & Co. Kg Methods of producing proteins in Chinese hamster ovary (CHO) cells
US7393923B2 (en) 2004-11-08 2008-07-01 The Regents Of The University Of California Red-shifted fluorescent proteins mPlum and mRaspberry and polynucleotides encoding the same
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US9758790B2 (en) 2004-12-08 2017-09-12 Ceres, Inc. Modulating the level of components within plants
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