WO2000034325A1 - Proteines fluorescentes d'especes anthozoaires non bioluminescentes, genes codant ces proteines et utilisation - Google Patents

Proteines fluorescentes d'especes anthozoaires non bioluminescentes, genes codant ces proteines et utilisation Download PDF

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WO2000034325A1
WO2000034325A1 PCT/US1999/029472 US9929472W WO0034325A1 WO 2000034325 A1 WO2000034325 A1 WO 2000034325A1 US 9929472 W US9929472 W US 9929472W WO 0034325 A1 WO0034325 A1 WO 0034325A1
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dna
fluorescent protein
isolated
encodes
organism
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PCT/US1999/029472
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Sergey Anatolievich Lukyanoy
Arcady Fedorovich Fradkov
Yulii Aleksandrovich Labas
Mikhail Vladimirovich Matz
Yu Fang
Wenyan Tan
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Clontech Laboratories, Inc.
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Publication of WO2000034325A1 publication Critical patent/WO2000034325A1/fr

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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 (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 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 b een 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.
  • the fluorescent protein has the amino acid sequence selected from the group consisting of SEQ ID Nos. 56 and 58.
  • 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 selected from the group consisting of SEQ ID Nos. 56 and 58.
  • 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, insect cells and yeast cells
  • 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) in codon sequence due to the degeneracy of the genetic code, and which encodes a fluorescent protein.
  • the protein has the amino acid sequence selected from the group consisting of SEQ ID Nos. 56 and 58.
  • 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, the 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., zFP538.
  • 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.
  • 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 the 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 o f the novel fluorescent protein from Zoanthus sp. , zFP538.
  • Figure 3 shows the excitation and emission spectrum of mutant zFP538, i.e., ml28v.
  • Figure 4 shows transient expression of pYNFP m l 28v-N l and pEYFP-Nl in 293 cells, respectively.
  • pYNFP ml28v-Nl Figure 4B
  • Figure 4 A shows as bright fluorescent intensity as pEYFP-Nl ( Figure 4 A) by fluorescent microscopy. The fluorescence showed is green
  • Figure 5 shows that fusion protein PKC- ⁇ YNFP (M l 28V) translocated from cytosol to the plasma membrane when cells were treated with PMA (Phorbol 12-Myristate 13-Acetate).
  • Figure 5A shows the result from control (without the treatment) and
  • Figure 5B shows the result from PMA-treated cells.
  • 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).
  • ⁇ FP refers to yellow fluorescent protein
  • ' ⁇ YFP' refers to enhanced yellow fluorescent protein
  • the term “NFP” refers to novel fluorescent protein
  • the term “YNFP” refers to yellow novel fluorescent protein
  • YNFP refers to zFP538.
  • conventional molecular biology, microbiology, and recombinant DNA techniques within the skill of the art. Such techniques are explained fully in the literature. See, e.g., Maniatis, Fritsch & Sambrook, "Molecular Cloning: A Laboratory Manual ( 1982) ; “DNA Cloning: A Practical Approach,” Volumes l and II (D.N. Glover ed . 1985); “Oligonucleotide Synthesis” (M.J. Gait ed.
  • 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 n o 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. The boundaries of the coding sequence are determined by a start codon a t the 5' (amino) terminus and a translation stop codon at the 3 ' (carboxyl) terminus.
  • 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 st 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 c u t 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 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 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.
  • 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.
  • 3552- 59 is used.
  • the present invention is directed to an isolated DNA selected from the group consisting of: (a) isolated DNA from an 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 the 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 and 57, and the fluorescent protein has the amino acid sequence selected from the group consisting of SEQ ID Nos. 56 and 58. More preferably, the DNA is zFP538 or M128V.
  • 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 selected from the group consisting of SEQ ID Nos. 56 and 58.
  • the vector is constructed by amplifying the DNA and then inserting the amplified DNA into EGFP-N1 backbone, or by fusing different mouse ODC degradation domains such as dl, d2 and d376 t o the C-terminal of the DNA and then inserting the 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
  • the organism is from Sub-class Zoantharia, Order Zoanthidea. More preferably, the 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 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 said DNA is from an organism from Class Anthozoa and wherein said organism does not exhibit bioluminescence;
  • the isolated and purified fluorescent protein is zFP538.
  • 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 as 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 green spots on oral disk
  • 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 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
  • 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 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)
  • Discosoma striata NGH NFP (SEQ ID No. 4) (SEQ ID No. 13)
  • 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 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, 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 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, 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 ampiciUin) at 37°C overnight. 100 ⁇ l of the overnight culture was transferred into 200 ml of fresh LB medium containing 100 ⁇ g/ml of ampiciUin and grown at 37°C, 200 rpm up to OD 600 0.6-0.7. 1 mM IPTG was then added to the culture an 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).
  • zFP538 One of the full-length cDNAs encoding novel fluorescent proteins is described herein (zFP538).
  • the nucleic acid sequence and deduced amino acid sequence are SEQ ID Nos. 55 and 56, respectively.
  • the spectral properties of zFP538 are listed in Table 7, and th e emission and excitation spectrum for zFP538 is shown in Figure 2.
  • ⁇ relative brightness is extinction coefficient multiplied by quantum yield divided by the same value for A. victoria GFP.
  • M128V was generated by introducing a wrong nucleotide in PCR during site-specific mutagenesis at position 65.
  • One bright yellow colony was obtained, and the sequence of this clone was performed. It showed that this clone contained wild type amino acid Lysine (K) at position 65, but had a substitution from Methionine (M) to Valine (V) at position 1 28 (numbering according to GFP).
  • K wild type amino acid Lysine
  • M Methionine
  • V Valine
  • SEQ ID No. 58 The nucleic acid sequence of M128V is shown in SEQ ID No. 57, and the deduced amino acid sequence is shown in SEQ ID No. 58.
  • M128V has spectral characteristics very similar to wild type protein zFP538 but folds much faster.
  • Figure 3 shows the emission and excitation spectrum for Ml 28V.
  • Table 8 lists the spectral properties of Ml 28V.
  • *relative brightness is extinction coefficient multiplied by qu antum yield divided by the same value for A. victoria GFP.
  • pYNFPwt showed less fluorescent intensity than EYFP (data not shown); however, pYNFPM128V showed as bright fluorescent intensity as EYFP by fluorescent microscopy ( Figures 4A and 4B).
  • destabilized zFP538 vectors were constructed by fusing different mou se ODC degradation domains such as dl and d2 to the C-terminal o f zFP538.
  • the dl version of destabilized YNFP has three E to A mutations within MODC degradation domain compared to d2 version.
  • Vectors pYNFPM128V-MODCdl and pYNFPM128V-MODCd2 were constructed in EGFP-N1 backbone.
  • M128V has fast folding and bright fluorescent intensity, which makes it useful for number of applications.
  • Some fusion proteins were tested such as PKC-gamma-YNFP (M128V).
  • PKC-gamma was observed to translocate from cytosol to the plasma membrane when cells were treated with PMA (Phorbol 12-Myristate 13-Acetate) ( Figures 5A and 5B).
  • Humanized M128V was generated, and then placed into th e pEGFP-Nl backbone.
  • This vector has the same mutiple cloning sites a s pEGFP-Nl . Construction of CI and pEGFP is in the process.

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Abstract

La présente invention concerne de nouvelles protéines fluorescentes provenant d'organismes anthozoaires non bioluminescents. L'invention concerne également des ADN complémentaires codant les protéines fluorescentes.
PCT/US1999/029472 1998-12-11 1999-12-10 Proteines fluorescentes d'especes anthozoaires non bioluminescentes, genes codant ces proteines et utilisation WO2000034325A1 (fr)

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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
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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
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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
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EP1135532A1 (fr) * 1998-12-11 2001-09-26 Clontech Laboratories Inc. Proteines fluorescentes appartenant a des especes non bioluminescentes de la classe des anthozoaires, genes codant pour ces proteines et utilisations
US7338784B2 (en) 1998-12-11 2008-03-04 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
EP2308974A3 (fr) * 1999-10-14 2011-08-10 Clontech Laboratories Inc. Chromo/fluoroprotéines dérivées d'anthozoaires et leurs procédés d'utilisation
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
US7858844B2 (en) 2001-02-21 2010-12-28 Clontech Laboratories, Inc. Non aggregating fluorescent proteins and methods for using the same
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US7183399B2 (en) 2001-10-12 2007-02-27 Clontech Laboratories, Inc. Nucleic acids encoding linked chromo/fluorescent domains and methods for using the same
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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
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