[go: up one dir, main page]

WO2000034321A1 - Proteines fluorescentes d'espece non bioluminescente de la classe des anthozoaires, genes codant pour ces proteines et utilisations - Google Patents

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

Info

Publication number
WO2000034321A1
WO2000034321A1 PCT/US1999/029402 US9929402W WO0034321A1 WO 2000034321 A1 WO2000034321 A1 WO 2000034321A1 US 9929402 W US9929402 W US 9929402W WO 0034321 A1 WO0034321 A1 WO 0034321A1
Authority
WO
WIPO (PCT)
Prior art keywords
dna
fluorescent protein
isolated
encodes
seq
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/US1999/029402
Other languages
English (en)
Inventor
Sergey Anatolievich Lukyanoy
Arcady Fedorovich Fradkov
Yulii Aleksandrovich Labas
Mikhail Vladimirovich Matz
Brigitte Angres
Gisele Green
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Takara Bio USA Inc
Original Assignee
Clontech Laboratories Inc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Clontech Laboratories Inc filed Critical Clontech Laboratories Inc
Publication of WO2000034321A1 publication Critical patent/WO2000034321A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

Links

Classifications

    • 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 o f 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 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 b y Davis et al. in Dev. Biology 170 (1995), 726-729.
  • GFP tertiary structure resembles a barrel (Orm ⁇ 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 a s 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 o f which has increased synthesis in mammalian cells (Haas, et al., Current Biology 6 ( 1996), 315-324; Yang, et al., Nucleic Acids Research 2 4 ( 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; an d (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 th at encodes a fluorescent protein.
  • the DNA is isolated from a non-bioluminescent organism from Class Anthozoa. More preferably, the DNA has the sequence shown in SEQ ID No. 55 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 the 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.
  • a representative example of bacterial cell is an E. coli cell.
  • 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) 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) i n 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 th e DNA is from an organism from Class Anthozoa and wherein th e 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 o f (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 Alcyonaria, Order Stolonifera. More preferably, th e organism is from Family Clavulariidae, Genus Clavularia. Most particularly, the present invention is drawn to a novel fluorescent protein from Clavularia sp. , cFP484.
  • 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.
  • FIG. 1 shows the modified strategy of 3'-RACE used t o isolate the target fragments. Sequences of the oligonucleotides u s ed 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 th e sequences of degenerate primers). In the case of Clavularia sp., th e first degenerate primer used was NGH (SEQ ID No. 4), and the second degenerate primer used was GEG(a) (SEQ ID No. 6).
  • Figure 2 shows the excitation and emission spectrum o f the novel fluorescent protein from Clavularia sp. , cFP484.
  • Figure 3 shows transient expression of cFP484 lacking th e
  • Figure 3 A shows the image emitted by ⁇ 19 cFP484 using the filter set XF 1 1 4 (Omega Optical).
  • Figure 3B shows phase contrast image taken from th e same field of view as that in Figure 3 A and overlayed with the image i n Figure 3 A.
  • 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.
  • SEQ ID No. 54 The sequence of Aequorea victoria GFP (SEQ ID No. 54) has been disclosed in Prasher et al., Gene 111 (1992), 229-33.
  • EGFP refers to mutant variant o f
  • GFP having two amino acid substitutions: F64L and S65T (Heim et al. , Nature 373 (1995), 663-664).
  • the term "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 Resea rch 24 ( 1996), 4592-4593).
  • 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 o f 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 th e 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, th at 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 .
  • promoters including inducible promoters, may be used t o drive the various vectors of the present invention.
  • the terms "restriction endonucleases” an d “restriction enzymes” refer to bacterial enzymes, each of which c u t double-stranded DNA at or near a specific nucleotide sequence.
  • a cell has been "transformed” or “transfected” b y 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 th e 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 n o 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 brou ght 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 an d whose product may be assayed easily and quantifiably when th e construct is introduced into tissues or cells.
  • the amino acids described herein are preferred to be in th e
  • 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 shown in SEQ ID No. 5 5 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 the 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 the vector of the present invention, which expresses a fluorescent protein of the present invention.
  • the cell is selected from the group consisting o f bacterial cells, mammalian cells, plant cells and insect cells.
  • a representative 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 th e DNA is from an organism from Class Anthozoa and wherein th e 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 o f (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 Alcyonaria, Order Stolonifera.
  • th e organism is from Family Clavulariidae, Genus Clavularia.
  • the present invention is also directed to an isolated and 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 the DNA is from an organism from Class Anthozoa and wherein the 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 cFP484.
  • 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 a s 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).
  • Discosoma sp Western Pacific orange-red spots oral disk
  • Discosoma sp Western Pacific green spots on oral disk
  • Amplified cDNA samples were then prepared a s 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 0.1 ⁇ 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 o f this dilution was used in subsequent procedures.
  • T7-TN3 5'-GTAATACGACTCACTATAGGGCCGCAGTCGACCG(T) 13
  • TS-primer 5'-AAGCAGTGGTATCAACGCAGAGT
  • T7-TS 5'-GTAATACGACTCACTATAGGGCAAGCAGTGGTATCAACGCAGAGT
  • PCR using degenerate primers was performed.
  • Degenerate primers were designed to match the sequence of the mRNAs in regions th at were predicted to be the most invariant in the family of fluorescent proteins. Four such stretches were chosen (Table 3) and variants o f 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 th e 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) PNAS 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, 4 0 sec.
  • the 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 th e manufacturer (CLONTECH), 200 ⁇ M dNTPs, 0.3 ⁇ M of the second degenerate primer (Table 4) and 0.1 ⁇ M of TN3 primer.
  • the cycling profile was (Hybaid OmniGene Thermocycler, tube control mode): 1 cycle for 95°C, 10 sec; 55°C (for GEG/GNG or PVM) or 52°C (for NFP), 1 min.; 72°C, 40 sec; 13 cycles for 95°C, l Osec; 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 th e 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 o f 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 o f 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 0.1 ⁇ M of TS primer (SEQ ID No.
  • Both primers had 5 ' -heels coding for a site for a restriction endonuclease; in addition, th e 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 th e amplified cDNA sample was added to a mixture containing l x Advantage KlenTaq Polymerase Mix with buffer provided by th e 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 to 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 LB medium containing 100 ⁇ g/ml of ampicillin and grown at 37°C, 200 rpm up to OD 600 0.6-0.7. 1 mM IPTG was then added to the culture a n d 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).
  • cFP484 One of the full-length cDNAs encoding fluorescent proteins found is described herein (cFP484).
  • the nucleic acid sequence an d deduced amino acid sequence are SEQ ID Nos. 55 and 56, respectively.
  • *relative brightness is extinction coefficient multiplied by quantum yield divided by the same value for A. victoria GFP .
  • ⁇ 19 cFP484 lacks the N-terminal first 19 amino acids o f cFP484, and ⁇ 38 cFP484 lacks the N-terminal first 38 amino acids of cFP484.
  • Mammalian expression vectors containing the DNA encoding the fluorescent protein ⁇ 19 cFP484 or ⁇ 38 cFP484 are generated, which are named as p ⁇ 19 NFP2-N1 and p ⁇ 38 NFP2-N1 , respectively.

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Biophysics (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Zoology (AREA)
  • Biochemistry (AREA)
  • Toxicology (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Medicinal Chemistry (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Peptides Or Proteins (AREA)

Abstract

La présente invention concerne des protéines fluorescentes d'organismes non-bioluminescents de la classe des anthozoaires. L'invention concerne aussi des ADNc codant pour ces protéines fluorescentes.
PCT/US1999/029402 1998-12-11 1999-12-10 Proteines fluorescentes d'espece non bioluminescente de la classe des anthozoaires, genes codant pour ces proteines et utilisations Ceased WO2000034321A1 (fr)

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
US21033098A 1998-12-11 1998-12-11
US09/210,330 1998-12-11
US41891799A 1999-10-15 1999-10-15
US09/418,917 1999-10-15

Publications (1)

Publication Number Publication Date
WO2000034321A1 true WO2000034321A1 (fr) 2000-06-15

Family

ID=26905060

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US1999/029402 Ceased WO2000034321A1 (fr) 1998-12-11 1999-12-10 Proteines fluorescentes d'espece non bioluminescente de la classe des anthozoaires, genes codant pour ces proteines et utilisations

Country Status (1)

Country Link
WO (1) WO2000034321A1 (fr)

Cited By (18)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
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
WO2003070952A1 (fr) * 2002-02-25 2003-08-28 Riken Proteine fluorescente
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
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
US7338782B2 (en) 1998-12-11 2008-03-04 Clontech Laboratories, Inc. Nucleic acids encoding chromophores/fluorophores and methods for using 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
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
US9758790B2 (en) 2004-12-08 2017-09-12 Ceres, Inc. Modulating the level of components within plants
US9834584B2 (en) 1998-12-11 2017-12-05 Takara Bio Usa, Inc. Nucleic acids encoding chromophores/fluorophores and methods for using the same

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE19718640A1 (de) * 1997-05-02 1999-07-22 Joerg Wiedenmann Anwendung eines orange-fluoreszierenden Proteins und weiterer farbiger Proteine und der zugehörigen Gene aus der Artengruppe Anemonia sp. (sulcata) Pennant, (Cnidaria, Anthozoa, Actinaria) in Gentechnologie und Molekularbiologie

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE19718640A1 (de) * 1997-05-02 1999-07-22 Joerg Wiedenmann Anwendung eines orange-fluoreszierenden Proteins und weiterer farbiger Proteine und der zugehörigen Gene aus der Artengruppe Anemonia sp. (sulcata) Pennant, (Cnidaria, Anthozoa, Actinaria) in Gentechnologie und Molekularbiologie

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
ANDERLUH ET AL.: "Cloning, sequencing and expression of equinatoxin II", BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS,, vol. 220, no. 2, 1996, pages 437 - 442, XP002924747 *
MACEK ET AL.: "Intrinsic tryptohan fluorescence of equinatoxin II, a pore-forming polypeptide from the sea anemone, Actinia equina L, monitors its interaction with lipid membranes", EUROPEAN JOURNAL OF BIOCHEMISTRY,, vol. 234, 1995, pages 329 - 335, XP002924749 *
MATZ ET AL.: "Fluorescent proteins from nonbioluminescent Anthozoa species", NATURE BIOTECHNOLOGY,, vol. 17, no. 10, October 1999 (1999-10-01), pages 969 - 973, XP002924748 *

Cited By (35)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7442522B2 (en) 1998-12-11 2008-10-28 Clontech Laboratories, Inc. Nucleic acids encoding chromo or fluorescent proteins and method for using the same
US7338782B2 (en) 1998-12-11 2008-03-04 Clontech Laboratories, Inc. Nucleic acids encoding chromophores/fluorophores and methods for using the same
US7344862B2 (en) 1998-12-11 2008-03-18 Clontech Laboratories, Inc. Nucleic acids encoding chromophores/fluorophores and methods for using the same
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
US8012682B2 (en) 1998-12-11 2011-09-06 Clontech Laboratories, Inc. Nucleic acids encoding chromophores/fluorophores and methods for using the same
US7537915B2 (en) 1998-12-11 2009-05-26 Clontech Laboratories, Inc. Nucleic acids encoding chromophores/fluorophores and methods for using the same
US7338783B2 (en) 1998-12-11 2008-03-04 Clontech Laboratories, Inc. Nucleic acids encoding chromophores/fluorophores and methods for using the same
US7338784B2 (en) 1998-12-11 2008-03-04 Clontech Laboratories, Inc Nucleic acids encoding chromophores/fluorophores and methods for using the same
US9834584B2 (en) 1998-12-11 2017-12-05 Takara Bio Usa, Inc. Nucleic acids encoding chromophores/fluorophores and methods for using the same
US7338785B2 (en) 1998-12-11 2008-03-04 Clontech Laboratories, Inc. Nucleic acids encoding chromophores/fluorophores and methods for using the same
US7442521B2 (en) 1998-12-11 2008-10-28 Clontech Laboratories, Inc. Nucleic acids encoding chromophores/fluorophores and methods for using the same
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
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
US7150979B2 (en) 2001-02-21 2006-12-19 Clontech Laboratories, Inc. Nucleic acids encoding non-aggregating fluorescent proteins and methods for using the same
US8431769B2 (en) 2001-02-21 2013-04-30 Clontech Laboratories, Inc. Non-aggregating fluorescent proteins and methods for using the same
US6969597B2 (en) 2001-02-21 2005-11-29 Clontech Laboratories, Inc. Nucleic acids encoding non aggregating fluorescent proteins and methods for using the same
US8093450B2 (en) 2001-02-21 2012-01-10 Clontech Laboratories, Inc. Non-aggregating fluorescent proteins and methods for using the same
US7858844B2 (en) 2001-02-21 2010-12-28 Clontech Laboratories, Inc. Non aggregating fluorescent proteins and methods for using the same
EP2284195A1 (fr) 2001-09-18 2011-02-16 Carnegie Institution Of Washington Protéines hybrides destinées à la détection d'analytes
US7183399B2 (en) 2001-10-12 2007-02-27 Clontech Laboratories, Inc. Nucleic acids encoding linked chromo/fluorescent domains and methods for using the same
US7375201B2 (en) 2002-02-25 2008-05-20 Riken Fluorescent protein
WO2003070952A1 (fr) * 2002-02-25 2003-08-28 Riken Proteine fluorescente
US7956172B2 (en) 2003-06-16 2011-06-07 Riken Fluorescent protein and chromoprotein
WO2004111235A1 (fr) * 2003-06-16 2004-12-23 Riken Proteine fluorescente et proteine pigment
JP4852676B2 (ja) * 2003-06-16 2012-01-11 独立行政法人理化学研究所 蛍光蛋白質及び色素蛋白質
US8207322B2 (en) 2003-06-16 2012-06-26 Riken Fluorescent protein and chromoprotein
JPWO2004111235A1 (ja) * 2003-06-16 2006-07-27 独立行政法人理化学研究所 蛍光蛋白質及び色素蛋白質
US9758790B2 (en) 2004-12-08 2017-09-12 Ceres, Inc. Modulating the level of components within plants
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
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

Similar Documents

Publication Publication Date Title
EP1135532B1 (fr) Proteines fluorescentes appartenant a des especes non bioluminescentes de la classe des anthozoaires, genes codant pour ces proteines et utilisations
WO2000034318A1 (fr) Proteines fluorescentes d'especes non bioluminescentes de la classe des anthozoaires, genes codant pour ces proteines et utilisations de ces proteines
WO2000034326A1 (fr) Proteines fluorescentes non bioluminescentes d'especes anthozoaires, genes codant ces proteines et leurs utilisations
AU783767B2 (en) Anthozoa derived chromophores/fluorophores and methods for using the same
WO2000034321A1 (fr) Proteines fluorescentes d'espece non bioluminescente de la classe des anthozoaires, genes codant pour ces proteines et utilisations
WO2000034322A1 (fr) Proteines fluorescentes d'especes non bioluminescentes de la classe des anthozoaires, genes codant pour ces proteines et leurs utilisations
WO2000034323A1 (fr) Proteines fluorescentes d'especes non bioluminescentes de la classe des anthozoaires, genes codant pour ces proteines et utilisations
WO2000034324A1 (fr) Proteines fluorescentes d'especes non bioluminescentes de la classe des anthozoaires, genes codant pour ces proteines et utilisations
RU2303600C2 (ru) Новые хромофоры/флуорофоры и способы их применения
AU2003208520B2 (en) Fluorescent protein from aequorea coerulscens and uses therof
US20020160473A1 (en) Far red shifted fluorescent proteins
AU2003208520A1 (en) Fluorescent protein from aequorea coerulscens and uses therof
AU2002211722A1 (en) Nucleic acids encoding stichodactylidae chromoproteins
US7358336B2 (en) Fluorescent protein from Montastraea cavernosa
JP2006501804A (ja) 急速に成熟する蛍光タンパク質およびその使用法
US7067645B2 (en) mmFP encoding nucleic acids, polypeptides, antibodies and methods of use thereof
NZ517548A (en) Non-bioluminescent Anthozoa derived chromophores / fluorophores and methods for using the same
AU2006200881A1 (en) Anthozoa derived chromophores/fluorophores and methods for using the same
AU2002361624A1 (en) Novel chromophores/fluorophores and methods for using the same

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): JP

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): AT BE CH CY DE DK ES FI FR GB GR IE IT LU MC NL PT SE

121 Ep: the epo has been informed by wipo that ep was designated in this application
122 Ep: pct application non-entry in european phase