[go: up one dir, main page]

WO1996027675A1 - Expression de la proteine fluorescente verte (pfv) de meduse dans des plantes - Google Patents

Expression de la proteine fluorescente verte (pfv) de meduse dans des plantes Download PDF

Info

Publication number
WO1996027675A1
WO1996027675A1 PCT/GB1996/000481 GB9600481W WO9627675A1 WO 1996027675 A1 WO1996027675 A1 WO 1996027675A1 GB 9600481 W GB9600481 W GB 9600481W WO 9627675 A1 WO9627675 A1 WO 9627675A1
Authority
WO
WIPO (PCT)
Prior art keywords
gfp
sequence
modified
die
expression
Prior art date
Application number
PCT/GB1996/000481
Other languages
English (en)
Inventor
James Phillip Haseloff
Sarah Hodge
Douglas Prasher
Kirby Siemering
Original Assignee
Medical Research Council
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 Medical Research Council filed Critical Medical Research Council
Priority to AU48843/96A priority Critical patent/AU4884396A/en
Publication of WO1996027675A1 publication Critical patent/WO1996027675A1/fr

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
    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8201—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation
    • C12N15/8209—Selection, visualisation of transformants, reporter constructs, e.g. antibiotic resistance markers

Definitions

  • This invention relates to improvements in gene expression, especially improvements in expression of the Green Fluoresecent Protein (GFP) gene, and to a method of detecting the presence and/or expression in a host of a gene of interest.
  • GFP Green Fluoresecent Protein
  • Proteins with high intrinsic fluorescence are involved in photosynthesis and bioluminescence, and in most cases possess a protein-bound chromophore.
  • the highly fluorescent phycobiliproteins require complex tetrapyrrole groups, and the blue and yellow fluorescent proteins from Vibrio fischeri must bind lumazine and flavin mononucleotide. respectively. This requirement for an external chromophore complicates the use of these proteins as reporters for gene expression.
  • the green fluorescent protein (GFP) from the jellyfish Aequorea victoria does not share this requirement for an external chromophore.
  • Aequorea victoria are brightly luminescent, with light appearing as glowing points around the margin of the jellyfish umbrella. Light arises from yellow tissue masses which each consist of about 6000-7000 photogenic cells (Davenpo ⁇ & Nichol, 1955 Proc. Roy. Soc. Ser. B 144. 399-411). The cytoplasm of these cells is densely packed wjth fine granules of about 0.2 ⁇ m diameter which are enclosed by a unit membrane and contain the components necessary for bioluminescence (Anderson & Cormier, 1973 J. Biol. Chem. 248, 2937-2943). The components include a Ca" activated photoprotein, aequorin, that emits blue-green light, and an accessory green fluorescent protein (GFP) which accepts energy from aequorin and re-emits it as green light.
  • GFP accessory green fluorescent protein
  • GFP is an extremely stable protein of 238 amino acids.
  • the fluorescent properties of the protein are unaffected by prolonged treatment with 6M guanidine HC1, 8M urea or 1 % SDS, and two day treatment with various proteases such as trypsin, chymotrypsin, papain, subtilisin, thermolysin and pancreatin at concentrations up to 1 mg/ml fail to alter the intensity of GFP fluorescence.
  • GFP is stable in neutral buffers up to 65 °C, and displays a broad range of pH stability from 5.5 to 12 (Bokman & Ward, 1981 Biochem. Biophys. Res. Comm. 101, 1372-1380).
  • the protein is intensely fluorescent, with a quantum efficiency of approximately 80% and molar extinction coefficient of around 4.5xl0 4 .
  • GFP absorbs light maximally at 395 nm and has a smaller absorbance peak at 475nm, and fluorescence emission peaks at 509nm, with a shoulder at 540nm (Morise et al., 1974 Biochemistry 13, 2656-2662).
  • researchers have successfully cloned and sequenced both the cDNA and genomic DNA sequences coding for A. victo ⁇ a GFP (Prasher et al. , 1992 Gene 111, 229-233).
  • the gfp gene contains at least three introns. and the sequences derived from the cDNA have been used for protein expression studies in Esche ⁇ chia coli, Caenorhabditis elegans (Chalfie et al. , 1994 Science 263. 802 et seq.) and Drosophila melanogaster (Wang & Hazelrigg, 1994 Nature 369, 400 et seq.). Fluorescent protein was produced in these different cell types and there appears to be little requirement for specific additional factors for post-translational modification of the protein, which may be autocatalytic or require common factors.
  • GFP has some advantages as a fluorescent reporter molecule, expression has been repo ⁇ ed to be problematic in some experimental systems (Cubitt et al. , 1995 Trends Biochem. Sci. 20, 448-455). Expression of GFP in mammalian cells has been described as highly variable (Rizutto et al. , 1995 PNAS 92, 11899-11903); Kaether & Gerdes 1995, FEBS Lett. 369, 267-271; Pines 1995, Trends Genet. 11, 326-327) often requiring a strong promoter and decreased incubation temperature for good results (Ogawa et al. , 1995 PNAS 92, 11899-11903).
  • the invention provides a DNA sequence encoding Green Fluorescent Protein (GFP), the sequence being modified relative to the wild type sequence so as to allow for more efficient expression in a plant cell of a functional GFP polypeptide.
  • GFP Green Fluorescent Protein
  • the modified sequence is capable of efficient expression in a dicotyledonous plant, such as Arabidopsis.
  • GFP GFP
  • polypeptide possessing many of the properties of the naturally occurring protein, and particularly exhibiting intrinsic fluorescence.
  • the polypeptide need not necessarily fluoresce in the "green" pan of the visible spectrum, as the fluorescence prope ⁇ ies of the polypeptide (including the wavelength of fluorescence) may be substantially altered by one or more mutations.
  • the GFP polypeptide will generally have substantially the amino acid sequence of the wild type protein (as disclosed by Prasher et al.), but will preferably comprise one or more amino acid differences defined, and discussed in greater detail, below.
  • the GFP-coding DNA sequence is advantageously modified so as to reduce the probability of an RNA sequence transcribed therefrom being subject to erroneous splicing in a plant cell.
  • the DNA sequence is conveniently modified so as to comprise a plurality of nucleotide substitutions relative to the wild type sequence, which substitutions serve to reduce, or preferably entirely prevent, excision from the transcribed RNA of the ponion corresponding to nucleotides 400-483 of the DNA sequence. It has surprisingly been found by the present inventors that this portion of the sequence tends to be recognised in plant cells (panicularly dicotyledenous plants) as an intron, which is therefore excised by splicing of the RNA.
  • the nucleotide substitutions in the DNA sequence serve to decrease the A/U% content of the transcribed RNA, which is believed to decrease the likelihood of the sequence being treated by a plant cell as representing an intron.
  • the substitutions particularly decrease the A/U% content of the region corresponding to nucleotides 400-483.
  • the nucleotide substitutions are such as to preserve the amino acid sequence of the encoded polypeptide substantially unchanged in the portion encoded by nucleotides 400-483.
  • Other substitutions may advantageously be made to decrease the similarity between the GFP RNA sequence and the plant intron recognition consensus sequence (see Figure 2).
  • RNA sequences capable of being transcribed from the modified DNA sequence i.e. RNA sequences transcribed from the modified DNA sequence, or an RNA having a sequence such that it could be synthesised by transcription from the modified DNA sequence.
  • the sequence will typically further comprise transcription and translation signals (e.g. promoters, enhancers) and/or localisation signals recognised in plants.
  • Localisation signals may direct the expressed polypeptide to: the nucleus (e.g. SV40 large T antigen localisation signal, particularly in combination with other polypeptide sequences, which have been found to increase the efficiency of the signalling); mitochondria (e.g. cytochrome C oxidase subunit IV); endoplasmic reticulum - ER (e.g.
  • GFP polypeptide may be highly desirable when expression occurs at high levels, so as to minimise possible toxicity to host cells.
  • the sequence may advantageously be further modified in accordance with the manner described in the prior art (e.g. as disclosed by Heim et al., 1994, 1995, or Delagrave et al. , 1995, as cited previously). Whilst in general the DNA sequence is modified in such a way as to preserve the wild type amino acid sequence, it has been found that amino acid changes at specific residues are in fact desirable. In particular, the sequence may be modified so as to comprise an amino acid substitution at one or both of amino acid residues 163 and 175. Changes at these positions are found to alter the characteristics of the polypeptide in an unexpected and favourable manner.
  • amino acid substitutions at residue 163 and/or 175 have favourable effects on the characteristics of the GFP polypeptide when expressed in many different host cells (e.g. bacterial, yeast etc.). and such substitutions may advantageously be included independently of any modification of the DNA sequence made for increased efficiency of expression in plants.
  • the invention provides a modified GFP polypeptide comprising an amino acid substitution relative to the wild type protein at residue 163 and/or 175. Substitution at either residue, in isolation, has surprisingly been found to increase the thermotolerance of the polypeptide. Maximal thermotolerance is obtained by causing substitution at both residues.
  • valine 163 is substituted by alanine, or by a related amino acid (i.e. those having an aliphatic side chain: glycine, leucine and isoleucine; or those having an aliphatic hydronyl side chain: serine and threonine).
  • a related amino acid i.e. those having an aliphatic side chain: glycine, leucine and isoleucine; or those having an aliphatic hydronyl side chain: serine and threonine.
  • serine 175 is substituted by glycine, or by a related amino acid (i.e. those having an aliphatic side chain: alanine, leucine and isoleucine; or those having an aliphatic hydroxyl side chain: serine and threonine).
  • a related amino acid i.e. those having an aliphatic side chain: alanine, leucine and isoleucine; or those having an aliphatic hydroxyl side chain: serine and threonine.
  • modified GFP polypeptide comprises substitutions at both residues, conveniently 163-*alanine and 175 ⁇ glycine.
  • the modified GFP may additionally comprise other sequence differences relative to the wild type protein, particularly in, or immediately adjacent to, residues 65-67 (which residues give rise to the chromophore).
  • nucleic acid sequence is useful for example, as a marker, or as a reporter gene, in a wide variety of host cells (e.g. mammalian, bacterial, fungal, yeast or plant cells).
  • host cells e.g. mammalian, bacterial, fungal, yeast or plant cells.
  • the nucleic acid sequence may be further modified for expression in a particular host cell.
  • the thermotolerant GFP-coding sequence is to be expressed in a plant cell it will conveniently be modified in accordance with the first aspect of the invention.
  • the invention provides a nucleic acid construct comprising a nucleic acid sequence in accordance with the first aspect of the invention.
  • the construct is preferably an expression vector, comprising one or more regulatory signals (such as promoters etc. ) and is preferably suitable for use in a plant cell.
  • the construct will desirably include one or more restriction endonuclease sites, suitable for the insertion into the construct of other nucleic acid sequences, which in a preferred embodiment may be inserted in frame with the sequence of the invention.
  • the invention also provides a host cell, conveniently a plant cell, into which has been introduced a sequence in accordance with the first aspect of the invention.
  • the invention thus provides a plant cell transformation vector comprising the sequence of the invention.
  • the invention provides a method of screening plant cells, comprising introducing into at least some of a plurality of plant cells a DNA construct comprising a sequence in accordance with the invention, maintaining the cells under suitable conditions for an appropriate length of time so as to allow expression of a modified GFP from the construct, and selecting those cells which exhibit GFP-mediated fluorescence.
  • Suitable conditions and “an appropriate length of time” are well known to those skilled in the art from standard texts.
  • me vector further comprises a sequence of interest which, preferably, is present in frame with the modified GFP-coding sequence.
  • the invention thus provides a method of detecting the expression in a plant of a sequence of interest, comprising causing the sequence of interest to be present in frame with a modified GFP-coding sequence in accordance widi the first aspect of the invention so as to form a modified GFP/sequence of interest fusion, introducing the fusion into a plant, and monitoring the fluorescence thereof.
  • GFP-mediated fluorescence is thus an indicator of expression of the sequence of interest.
  • the invention provides a nucleic acid construct comprising a sequence in accordance with the second aspect of the invention.
  • the nucleic acid construct will desirably have many of the features of the nucleic acid construct in accordance with the third aspect of the invention. It will be apparent however that the construct may be useful in many different types of host cell, and may be constructed accordingly.
  • Figure 1A shows the sequences introduced, via PCR, flanking the GFP-coding sequence
  • Figure IB is a confocal micrograph of transformed yeast cells expressing GFP
  • Figure 2 A is a photograph showing agarose gel electrophoresis analysis of PCR products
  • Figure 2B is a schematic illustration of the portion of DNA not represented in the mis- spliced mRNA produced in plants from the wild type GFP-coding sequence;
  • Figure 3A is a photograph showing the DNA sequence determination of the reverse transcript produced from mis-spliced mRNA
  • Figure 3B is a comparison between a portion of the GFP wild type sequence and a plant intron consensus sequence
  • Figure 4 shows a comparison of part of the wild type A. victoria GFP sequence with a modified GFP-coding sequence in accordance with the invention
  • Figure 5 is a series of confocal micrographs (at different magnification) showing parts of a plant expressing a modified GFP-coding sequence in accordance with the invention:
  • Figure 6 shows a comparison of pan of three modified GFP-coding sequences in accordance with the invention, together with the amino acid sequences encoded thereby;
  • Figure 7 is a graph of relative fluorescence (arbitary units) against time (minutes) for E. coli strains expressing modified GFP (open squares) or modified, mutated GFP (filled circles);
  • Figure 8 is a photograph of a Western blot, probed with anti-GFP antibody
  • Figure 9 is a bar chart showing the amount of fluorescence associated with cultures expressing modified GFP (open columns) on modified, mutated GFP (shaded columns) incubated at four different temperatures;
  • Figure 10 is a graph of fluorescence against time (minutes) for yeast cultures at 25 °C or 37°C, the cultures having been grown initially in anaerobic conditions, with oxygen introduced at time zero;
  • Figure 11 is a picture of a Western blot showing expression of modified GFP or modified, mutated GFP (GFP A) by E. coli cultures at 25 or 37°C, with comparison between soluble and insoluble culture fractions;
  • Figure 12 is a graph of absorbance against wavelength for soluble (filled circles) or insoluble (open circles) GFP;
  • Figure 13 is a picture of yeast cultures, grown at 25 or 37 °C and expressing modified GFP, or modified, mutated GFP (GFP A);
  • Figure 14 is a graph of fluorescence against wavelength (nm), showing the excitation spectra (squares) and emission spectra (circles) respectively, of modified GFP (solid lines) and two mutated forms of modified GFP, GFPA (dashed lines) and GFP5 (dotted lines);
  • Figure 15 is a comparison of the nucleotide sequence of wild-type gfp and a modified gene m-gfp5, and the polypeptides encoded thereby. Nucleotide sequence differences are shown in bold. The m-GFP5 amino acid sequence is shown beneath the nucleotide sequence. The three amino acid differences between the encoded polypeptides are indicated;
  • Figure 16 shows the sequence of another modified m-gfp gene, termed m-gfp5-ER, and the amino acid sequence of the polypeptide encoded thereby;
  • Figure 17 shows a number of confocal micrographs (A-H) of Arabidopsis seedlings expressing modified gfp genes in accordance with the invention.
  • a synthetic gfp gene was constructed using the polymerase chain reaction (PCR).
  • the plasmid pGFPlO. l (described by Prasher et al. , 1992 Gene 111, cited above) contains a cloned A. victoria gfp cDNA, and was used as template for PCR amplification (with Thermococcus litoralis Vent polymerase) with synthetic oligomer primers which were used to incorporate new sequences flanking the GFP coding sequence.
  • the sequence of the primer oligonucleotides was:
  • GGCC ATCCAAC ⁇ AGATATAACAATGAGTAAAGGAGAAGAACTTTTCACT (Seq. ID No. 1) and GGCGAGCTCTTATTTGTATAGTTCATCCATGCC (Seq. ID No. 2).
  • Figure 1A The newly-incorporated sequences are shown in Figure 1A.
  • the sequence existing in pGFPlO. l is shown italicised.
  • the added sequences are shown in normal type. These included: recognition sites for the restriction endonucleases BamHl and acl placed at the 5' and 3' termini of the amplified fragment: a Shine-Delgamo ribosome binding site (RBS) sequence positioned upstream of the initiation codon to ensure efficient translation of the transcribed gene in E. coli, and the sequence AAC A inserted between positions -4 and -1 for efficient translation in plants.
  • RBS Shine-Delgamo ribosome binding site
  • the PCR-amplified fragment was subcloned into pUC119 for bacterial expression, and into an episomal yeast plasmid vector, pVT103-U (Vernet et al. , 1987 Gene 52, 225-233) which contains a yeast 2 ⁇ M origin of replication and a truncated form of the yeast ADH1 promoter to allow high level expression of the cloned GFP gene in Saccharomyces cerevisiae.
  • S. cerevisiae MGLD-4a (a, leu2, ura3, his3, trpl, lys2) cells were transformed using the lithium acetate method described by Ito et al. (1983).
  • gfp is mis-spliced in Arabidopsis
  • the successful expression of GFP in Arabidopsis requires proper production of the apoprotein, before post-translational modification to form the chromophore.
  • the inventors therefore used PCR-based methods to verifv the correct insertion of the 35S promoter-driven gfp cDNA. and to check mRNA transcription and processing in transformed plantlets.
  • Nucleic acids were extracted from plantlets and either treated with RNase, or DNase treated and reverse transcribed using oligo(dT) g primer.
  • the gfp sequences in these extracts were therefore derived from genomic DNA or transcribed mRNAs, respectively.
  • the gfp sequence was PCR-amplified from these separate extracts and products were analysed by restriction endonuclease digestion, as shown in Figure 2A.
  • RNA RNase-treated DNA sample
  • DNase-treated/ reverse transcribed sample is shown on the right of each pair.
  • the samples were either loaded onto gel without prior restriction ("uncut”, extreme left hand pair of samples) or loaded after prior digestion with (from left to right): Ncol; Rsal; Dral; Accl; Hindi; or Avail. It can be seen that whilst the expected product was obtained after amplification of the gene, RT-PCR of mRNA sequences gave rise to a truncated product.
  • the shortened RT-PCR product was cloned and sequenced (Fig. 3A), and a deletion of 84 nucleotides between residues 400-483 was located.
  • the nucleotide sequences bordering this deletion are shown in figure 3B, and demonstrate similarity to known plant introns.
  • the sequence across the splice site (marked with an anow in Figure 3A) thus reads (5 * to 3') ...AG/AC... .
  • Matches were found for important residues at the 5' and 3' splice sites (reviewed by Luefrsen et al.
  • the jellyfish gene was mutated to produce a modified gfp (m-gfp) suitable for expression in Arabidopsis, as described below.
  • GTCTCCCTCAAACTTGACTTC (Seq. ID No. 4).
  • the oligonucleotides were purified by electrophoresis in a 5% polyacrylamide gel containing TBE and 7M urea. The gel was stained briefly with 0.05% toluidine blue, and the full-length oligonucleotides were excised, and eluted overnight in 0.5M ammonium acetate, O. lmM Na ; EDTA, 0.1 % SDS.
  • the oligonucleotides share 17 nucleotides of complementarity at their 3' termini, and were annealed and elongated after several rounds of thermal cycling with Vent polymerase. The extended product was cloned between the Nde I and Ace I sites of gfp.
  • mutant clones were screened for the presence of the diagnostic restriction endonuclease sites, Cla I, Ava II, and the desired fragment (m-gfp) was subcloned into M13 and its sequence verified by DNA sequencing using me dideoxynucleotide chain termination technique with T7 DNA polymerase.
  • the modifications introduced by the synthetic oligonucleotides were intended to alter the sequences which might be involved in 5' splice site recognition and to decrease the A:U content of the putative intron, as shown in Figure 4.
  • the upper DNA sequence is that of a portion of the wild type A. victoria GFP.
  • the lower DNA sequence is that of a portion of a modified GFP-coding sequence.
  • the corresponding amino acid sequence is shown beneath the DNA sequences.
  • Modified nucleotides are shown outlined. All DNA modifications affect only codon usage, and the m-g p-encoded amino acid sequence is identical to that of the wild-type jellyfish polypeptide.
  • the "pseudo- intron" sequence is underlined and the cryptic splice junctions are arrowed. Nucleotide and amino acid residue numbering (on the left and right, respectively, of the oblique stroke) start from the initiation codon.
  • the boxed hexanucleotide sequences are Ndel and Accl recognition sites respectively.
  • the m-gfp sequence was inserted behind the 35S promoter in pBI121 , and introduced into Arabidopsis using the root transformation technique.
  • Brightly green fluorescent cells were seen after co-cultivation with Agrobacterium. As shoot regeneration progressed, explants with different levels of green fluorescence could be observed. Regenerating callus and shoots develop a bright red autofluorescence due to the formation of chlorophyll within the tissues, and with the brightest m-gfp transformants the green fluorescence was clearly detectable against this autofluorescent background using a hand held UV lamp. This was similar to the levels of green fluorescence seen in transformed yeast and E. coli. However, these very bright Arabidopsis transformants regenerated and set seed rather poorly. Nevertheless, seeds were obtained from over 50 transformed lines, allowed to germinate, and screened by epifluorescence microscopy. Several of the brightest lines were used for confocal laser scanning microscopy.
  • the fluorescence properties of GFP and chlorophyll allow the use of fluorescence microscopes equipped with common filter sets for fluorescein and rhodamine for dual imaging in plant cells. Intact five day old m-g/p-transformed Arabidopsis seedlings were mounted in water for confocal laser scanning microscopy. GFP fluorescence could be clearly visualised in the transformed tissues, and chloroplasts provided a very effective counter fluor in the upper parts of the plant. Optical sectioning of the m-gfp transformed plants gives selective visual access to the internal details of living plant structure, as shown in Figure 5. without any need for staining or dissection. For e;xample.
  • GFP is found throughout the cytoplasm, but appears to accumulate within the nucleoplasm. It appears excluded from vacuoles, organelles and other bodies in the cytoplasm, and is excluded from the nucleolus. Similarly, in optical sections of cotyledon and hypocotyl tissues. GFP is found throughout the cytoplasm and nucleoplasm. The relationship of cells within the tissues is clearly discernible.
  • GFP fluorescence allows visualisation of trans-vacuolate cytoplasmic threads, and the thin cytoplasmic strands which underly the cell wall and which may be aligned with cytoskeletal elements. The movement of organelles through cytoplasmic streaming could also be observed in these living cells.
  • the sequence of m-gfp was mutated by PCR in the presence of limiting nucleotide concentrations.
  • the template plasmid was pBSm-gfp4, a derivative of TU#65 (Chalfie et al., 1994 Science 263, 802-805) in which gfp has been replaced with m-gfp.
  • the primers used were the T3 and T7 primers (New England Biolabs) that are complementary to the flanking T3 and T7 promoters present in the vector sequence.
  • the mutant library thus obtained was transformed into E. coli strain XLl-Blue (Stratagene) and incubated overnight at 37 °C on TYE agar containing 50 ⁇ g/ml ampicillin and 1 mM IPTG. Colonies were illuminated with a long wavelength UV lamp (UVP Model B 100 AP) and visually screened for increased fluorescence.
  • UVP Model B 100 AP UV lamp
  • the coding regions of two of the brightest mutant genes (m-gfpA and m-gfpB) thus identified, as well as that of m-gfp, were amplified by PCR (30 cycles of 1 min at 94°C.
  • the positions of the mutations responsible for the bright phenotypes of m-gfpA and m- gfpB were then localised by recombination of the muumt genes with m-gfp .
  • the pUC 119 derivatives containing m-gfp A and m-gfpB were cleaved with either BamHl and Ncol, Ncol and Clal, or Clal and Sad.
  • the restriction fragments were gel purified and ligated to the m-gfp pUC119 derivative that had been cleaved with the same combination of enzymes and gel purified.
  • the sequence of the m-gfp gene was modified so as to code for the VI 63 A and S175G substimtions described above and the I167T substimtion described in the prior art (1994 Heim et al. , Proc. Natl. Acad. Sci. USA 91. 12.501-12.504, which substitution inverts the ratio of the 400-475 nm excitation peaks), as well as to further alter the codon usage of the gene in order to eliminate potential plant intron sequences generated by the introduction of these mutations.
  • the sequence differences between this modified gene termed, m-gfp5, and the original gfp gene, and their respective polypeptides are summarised in Fig. 15 (Seq. ID Nos. 5-8).
  • the m-gfp5 gene was constructed by PCR amplification (30 cycles of 30 sees at 94 °C, 30 sees at 55 °C and 30 sees at 72 °C using VENT DNA Polymerase) of m-gfp using mutagenic primers.
  • the forward primer was an oligo corresponding to nucleotides 445- 560 of the m-gfp5 coding sequence shown in Figure 15 and the reverse primer was GFP- 3 'Sac.
  • the amplified fragment was cleaved and exchanged with the Accl-Sacl fragment of m-gfp to create m-gfp5.
  • BamHl-Sacl PCR fragments containing the m-gfp, m- gfpA and m-gfp5 genes were cloned downstream of the tac promoter of the expression vector pSE380 (Invitrogen), to give the plasmids pSE-GFP, pSE-GFPA and pSE-GFP5, respectively.
  • Expression from the tac promoter of pSE380 is tightly regulated due to the presence on the plasmid of the laclq gene.
  • yeast expression the same PCR fragments containing the m-gfp, m-gfp A and m-gfp5 genes were inserted downstream of the constitutive ADH1 promoter of pVT103-U (Vernet et al. , 1987), a yeast multicopy episomal plasmid containing the URA3 selectable marker.
  • the resulting plasmids were pVT-GFP, pVT-GFPA and pVT-GFP5. respectively.
  • amino acid substitutions present in m-GFPA suppress the temperature-sensitivity of GFP maturation
  • the post-translational mamration of GFP to the fluorescent form involves a number of steps.
  • the first step presumably, is folding of the apoprotein into a catalytic conformation that facilitates the novel reactions involved in formation of the chromophore.
  • These reactions consist of cyclisation and oxidation of the tripeptide Ser65-Tyr66-Gly67 to give a p-hydroxybenzylidene-imidazolidinone structure.
  • the chromophore Once the chromophore has been formed, it is then only fluorescent once GFP has adopted a fold which protects it from solvent effects. In principle, any of these processes could be sensitive to temperamre and thus be responsible for the observed thermosensitivity of GFP mamration.
  • FIG. 10 shows the rate of fluorescence development for cultures expressing modified GFP at 25°C (crosses) or 37°C (triangles), or cultures expressing GFPA at 25 °C (squares) or 37 °C (circles).
  • the time constant measured for the oxidation of m-GFP at 37°C was found to be approximately 3-fold faster than that measured at 25°C (16.2 ⁇ _ 0.3 min), indicating mat the post-translational oxidation of the GFP chromophore is not the step responsible for the temperamre sensitivity of mamration.
  • the time constants derived for m-GFPA at both 25°C and 37°C (22.5 ⁇ _ 1.4 min and 18.1 ⁇ _ 0.4 min. respectively) were actually slower than those measured for m- GFP.
  • cells containing pSE-GFP or pSE-GFPA were grown in 1.5 ml of 2xTY broth to an absorbance of 0.2 at 600 nm and then induced overnight with 0.2 mM IPTG.
  • the cultures were centrifuged at 13,000 rpm for 2 min, resuspended in 500 ⁇ l 50 mM Tris-HCl (pH 8.0), 2 mM EDTA, 100 ⁇ g/ml lysozyme, 0.1 % Triton X-100 and incubated at 30°C for 15 min.
  • the inventors made use of the characteristic absorption of the GFP chromophore in either the mature (Ward & Bokman, 1982 Biochemistry 21, 4535-4540) or chemically reduced state (Inouye & Tsuji 1994 FEBS Lett. 351, 211-214). If the aggregating species has already undergone the cyclisation reaction, GFP isolated from inclusion bodies should show this characteristic absorption. To facilitate the purification of protein for absorbance measurements, the inventors fused a polyhistidine tag to the C- terminus of m-GFP.
  • Histidine-tagging was achieved by die addition of 6 histidine codons to d e 3' ends of die modified gfp genes by PCR.
  • the genes were amplified using 5'Bam-GFP as the forward primer and the oligo
  • soluble and insoluble fractions of cells containing pSE-GFPHis grown at 25 °C and 37°C, respectively, were prepared as described previously.
  • GFP was purified from the fractions on Ni-chelate columns using the Ni-NTA Spin Kit (Qiagen). Purification from the soluble fraction was carried out according to the protocol for the purification of histidine- tagged proteins under native conditions. After clearance of cellular debris from the insoluble fraction by centrifugation at 13.000 rpm for 30 min. purification was carcied out according to the protocol for purification of histidine-tagged proteins under denaturing conditions, except that d e protein was eluted with resolubilisation buffer containing 250 mM imidazole.
  • cells were grown in 100 ml of 2xTY broth at 37 °C to an absorbance of 0.2 at 600 nm and then induced overnight with 0.5 mM IPTG. Cells were harvested by centrifugation at 6,000 rpm for 10 min and lysed by resuspension in 4 ml 20 mM Tris-HCl (pH 7.9), 500 mM NaCl, 5 mM imidazole, 0.1 % sarkosyl, 0.1 % deoxycholate. 2.25 M Guanidine-HCl.
  • Nucleic acids were precipitated by d e addition of 5ml isopropanol and removed by centrifugation at 10,000 rpm for 10 min. Fluorescent histidine-tagged proteins were purified from the supernatant on Ni-chelate columns (Qiagen) and eluted with 2ml of 20mM Tris-HCl (pH 7.9), 500 mM NaCl, 150 mM imidazole. For all purifications, protein purity was assayed by SDS-PAGE and found to be >95%. Protein concentrations were determined by Bradford assay (Bio-Rad Protein Assay kit) using bovine serum albumin as a standard.
  • Absorbance spectra were recorded on a Cary 3 UV-Visible Spectrophotometer (Varian) at 25 °C. The optical pathlength was 1 cm. Fluorescence spectra were recorded on a Hitachi F-4500 fluorimeter at 25°C using 4mm/10mm cuvettes. The bandpass for both the excitation and d e emission monochromators was 5 nm, the scan speed 240 nm per min and the response time automatically adapted by die device. All spectra were corrected following the supplier's procedure for calibration of the fluorimeter using Rhodamine-B as standard. Emission spectra were recorded at a fixed wavelength of the excitation maximum, excitation spectra at a fixed wavelength of the emission maximum.
  • thermosensitivity of m-GFP mamration observed in the yeast Saccharomyces cerevisiae is also a result of the thermosensitivity of apoprotein folding, it should be suppressed by me substimtions present in m-GFPA.
  • me substimtions present in m-GFPA die inventors incubated strains of cerevisiae containing either pVT-GFP or pVT-GFPA on agar plates at either 25°C or 37°C.
  • the substimtions present in m-GFPA also suppress the thermosensitivity of m-GFP expression in yeast. This result indicates diat the temperature-dependent mis-folding of the m-GFP apoprotein is not simply an artefact of an £. coli overexpression system, but is also the basis for the thermosensitivity of m- GFP mamration in a heterologous eukaryotic system.
  • Fluorescence spectroscopy of purified histidine-tagged m-GFP and m-GFPA revealed mat the fluorescence spectra of m-GFPA are essentially unchanged from those of m-GFP except for a decrease in the amplitude of the 475 nm excitation peak relative to the amplitude of the 400 nm excitation peak (Fig. 14). Aldiough this spectral change is advantageous for applications which utilise 400 nm excitation, it is also detrimental for those which utilise 475 nm excitation.
  • the ideal spectral variant would be a protein which could be efficiently excited at either of these wavelengths. This characteristic would afford greater flexibility with regard to the range of applications in which the protein could be used.
  • m-GFP5 Histidine-tagged m-GFP5 was purified and its excitation and emission spectra analysed by fluorescence spectroscopy. As can be seen in Fig. 14, m-GFP5 has two excitation peaks (maxima at 395 nm and 473 nm) of almost exactly equal amplimde and an emission spectrum largely unchanged from that of m-GFP.
  • bacterial cells containing pSE-GFP or an expression plasmid containing m-gfp5 induced widi IPTG for 5 hours at 37°C. The fluorescence ( ⁇ e .
  • mgfp5 Further modification of mgfp5 was achieved. Two synthetic oligonucleotides were made, to act as mutagenic PCR primers to add an in-frame Ec ⁇ RI site at the 5' end of the gene and to add a sequence coding for the amino acid tag HDEL at the C terminal of the protein (which tag acts as an endoplasmic reticulum localisation signal).
  • the PCR mutagenised sequence was men used in a three-way ligation reaction with BamHl/ Sad - cut vector and a pair of synthetic oligonucleotides with fi ⁇ mHI/Ec ⁇ RI ends.
  • the synthetic oligos had die sequences:
  • the oligos were annealed, extended with Klenow polymerase and cut with BamHl/Eco .
  • the nucleotide sequence of d e resulting modified gene (m-gjp5-ER), and die amino acid sequence of its polypeptide product (Seq ID No.s 10 and 11 respectively), are shown in Figure 16.
  • the nucleotides encoding me signal sequence are shown in upper case letters, whiolst the rest of the sequence is in lower case letters.
  • the C terminal HDEL tag on the protein is apparent.
  • the modified gene when expressed in Arabidopsis, gave highly efficient concentration of GFP-mediated fluorescence in the endoplasmic reticulum (Figure 17).
  • the panels illustrate confocal micrographs of 5-day old A. thaliana seedlings expressing m-GFP (panels A-D) or m-GFP5-ER (panels E-H), imaged at 395nm excitation wavelengdi.
  • the GFP apoprotein must be produced in suitable amounts wi in the plant cells.
  • the fluorescent protein may need to be suitably targeted within the cell, to allow efficient post-translational processing, safe accumulation to high levels, or to allow easier distinction of expressing cells.
  • the inventors have shown that expression of the jellyfish gfp cDNA in Arabidopsis is curtailed by aberrant splicing, with an 84 nucleotide intron being efficiently excised from within the GFP coding sequence.
  • the recognition of introns in plant pre-mRNAs primarily requires conserved sequences found adjacent to die 5' and 3' splice sites, which are related to those found in other eukaryotes, and, atypically, a high A:U content within the intron.
  • the inventors altered potential recognition sequences at e 5' splice site, and decreased d e A:U content of the cryptic intron by in vitro mutagenesis to produce a modified m-gfp gene which was successfully expressed in transgenic Arabidopsis plants. It is likely that this m-g p gene will be useful for expression studies in other plants, which appear to share similar feamres involved in intron recognition.
  • introns found in yeast possess a requirement for conserved sequences located at the branch point, and introns found in animal cells (including jellyfish) share a conserved polypyrimidine tract adjacent to die 3' splice site. The lack of these additional feamres may allow correct processing of the gfp mRNA in fungal and animal cells.
  • GFP is a source of fluorescence-related free radicals, for example, it might be advisable to target the protein to a more localised compartment within die plant cell. Appropriate localisation signals are known to those skillled in die art and it should prove possible to incorporate these into die GFP polypeptide widiout unduly disrupting die fluorescence characteristics of the protein.
  • the inventors have adapted die green fluorescent protein (GFP) of Aequoria victoria for use as a genetic marker in Arabidopsis thaliana.
  • Transcripts of the jellyfish GFP coding sequence are mis-spliced in Arabidopsis, with an 84 nucleotide intron being efficiently excised.
  • a modified version of me gfp sequence has been constructed to destroy this cryptic intron, and to restore proper expression of the protein in plant cells.
  • GFP is mainly localised wid in the nucleoplasm and cytoplasm within transformed Arabidopsis cells, and its presence allows optical sectioning of intact plants using confocal laser scanning microscopy.
  • the modified gfp sequence may be useful for directly monitoring gene expression and protein localization at high resolution, and as a simply scored genetic marker in living plants.
  • m-gfp A major use for m-gfp would be as a replacement for the /3-glucuronidase gene, used as a reporter for promoter and gene fusions in transformed plants. Histochemical staining is used to identify cells expressing the GUS gene product, but a fluorescent product can be imaged directly and rapidly. Gene expression and protein localization can be observed in physiologically active cells without a prolonged and lethal staining procedure, and fluorescence microscopy techniques allow the high resolution imaging of GFP-expressing cells. In addition, it becomes feasible to follow dynamic events in living cells and tissues. 29
  • thermotolerant mutants the native apoprotein or one of its folding intermediates is thermodynamically unstable and the protein aggregates when in the unfolded state.
  • the substimtions present in the thermotolerant mutants could suppress the characteristic either by increasing the thermodynamic stability of the unstable species or by decreasing its steady state level by increasing the rate of chromophore cyclisation.
  • higher temperamres can allow proteins to overcome the thermodynamic barriers to the formation of off-pathway folding intermediates which may become kinetically trapped by aggregation. It is possible, therefore, that the substimtions present in the heat-tolerant mutants act by suppressing such a phenomenon by directing folding along the correct pad way at elevated temperamres.
  • the inventors have shown that oxidation of die GFP chromophore does not contribute to the temperamre sensitivity of mamration by measuring the reaction rate in yeast cells at both 25°C and 37°C (Fig. 3).
  • An interesting point arising from this experiment is that the time constants derived for m-GFP at both 25°C and 37°C (5.9 ⁇ 0.1 min and 16.2 0.3 min. respectively) are significantly faster than the 120 min estimated for the oxidation of GFP in bacteria by Heim et al. This observation may reflect a difference in the physiological states of yeast and bacterial cells following anaerobic growth or perhaps die presence of a catalysing factor in yeast cells.
  • the factor which limits how quickly fluorescent can be observed following protein synthesis may be the efficiency with which the apoprotein folds rather than die time taken for oxidation of the chromophore.
  • GFP isolated directly from the Aequorea jellyfish it would appear that more than half of the recombinant GFP in a soluble fraction does not have a chromophore.
  • these mutant proteins are much less fluorescent than GFP, a phenomenon which has been attributed to diem having sub-optimal extinction coefficients and/or quantum yields due to d e poor fit of the alternative amino acids into me central cavity normally occupied by d e tyrosine residue. It is possible, however, diat die observed low fluorescence of these mutants is due to detrimental effects of die substimtions on folding and/or chromophore formation, resulting in the presence of large amounts of non- fluorescent protein in soluble fractions. Therefore, it is feasible diat die proper mamration of these mutants might be enhanced by die introduction of the amino acid substimtions present in m-GFPA or m-GFP5.
  • m-GFP As well as in E. coli, mamration of m-GFP appears to be thermosensitive in die yeast Saccharomyces cerevisiae and in mammalian cells. Therefore, it appears that die sensitivity of apoprotein folding to temperamre may be a ubiquitous phenomenon. Indeed, it is interesting to note that the brightness of m-GFP in Arabidopsis thaliana is markedly increased by its retention in the endoplamic reticulum (see accompanying paper), where a high concentration of chaperonins may enhance proper folding. It is unlikely, however, that the folding defect of m-GFP would manifest itself in the same way in systems where lower expression levels mean that aggregation may not occur to the same extent as in an E. coli overexpression system.
  • thermotolerant mutants described here should result in improved expression in a wide range of experimental systems. Indeed, in diis work the inventors have demonstrated that die substimtions present in m-GFPA are capable of suppressing the thermosensitivity of m-GFP expression in the yeast Saccharomyces cerevisiae.
  • m-GFPA has also been observed to give rise to significandy increased fluorescence in Drosophila melanogaster embryos incubated at 25 °C (A Brand, personal communication) and expression of m-GFP5 fused to endoplasmic reticulum retention signals has been observed to result in high levels of fluorescence in Arabidopsis th ⁇ liana (data not shown).
  • expression of both m-GFPA and m-GFP5 has been found to result in greatly increased levels of fluorescence in mammalian cells. Therefore, we anticipate that the diermotolerant mutants described in this work and spectral variants derived from them will be of great benefit for expression in many experimental systems, particularly those such as mammalian cells that utilise higher incubation temperamres.
  • GTC CCA A ⁇ c ⁇ G ⁇ GAA ⁇ A GAT GGT GAT GTJ AAT GGG CAC AAA r 144

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Organic Chemistry (AREA)
  • Biotechnology (AREA)
  • Biomedical Technology (AREA)
  • Zoology (AREA)
  • Molecular Biology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Wood Science & Technology (AREA)
  • Biophysics (AREA)
  • General Health & Medical Sciences (AREA)
  • Biochemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • Toxicology (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Cell Biology (AREA)
  • Physics & Mathematics (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Plant Pathology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Microbiology (AREA)
  • Medicinal Chemistry (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Peptides Or Proteins (AREA)

Abstract

L'invention concerne une séquence d'ADN codant pour la protéine fluorescente verte (PFV), cette séquence étant modifiée par rapport à la séquence native pour permettre une expression plus efficace d'un polypeptide fonctionnel de la PFV dans une cellule végétale. On décrit également un polypeptide modifié de la PFV présentant des substitutions d'acides aminés par rapport à la séquence native, ce polypeptide modifié présentant des caractéristiques utiles lorsqu'il est produit dans différentes cellules hôtes.
PCT/GB1996/000481 1995-03-06 1996-03-04 Expression de la proteine fluorescente verte (pfv) de meduse dans des plantes WO1996027675A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU48843/96A AU4884396A (en) 1995-03-06 1996-03-04 Jellyfish green fluorescent protein (gfp) expression in plants

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
GB9504446.7 1995-03-06
GBGB9504446.7A GB9504446D0 (en) 1995-03-06 1995-03-06 Improvements in or relating to gene expression

Publications (1)

Publication Number Publication Date
WO1996027675A1 true WO1996027675A1 (fr) 1996-09-12

Family

ID=10770703

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/GB1996/000481 WO1996027675A1 (fr) 1995-03-06 1996-03-04 Expression de la proteine fluorescente verte (pfv) de meduse dans des plantes

Country Status (3)

Country Link
AU (1) AU4884396A (fr)
GB (1) GB9504446D0 (fr)
WO (1) WO1996027675A1 (fr)

Cited By (54)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1997040178A1 (fr) * 1996-04-19 1997-10-30 Mycogen Plant Science, Inc. SURVEILLANCE DE PLANTES TRANSGENIQUES A MARQUEURS $i(IN VIVO)
WO1997041228A3 (fr) * 1996-05-01 1997-12-11 Pioneer Hi Bred Int Utilisation de la proteine fluorescente verte comme marqueur de criblage pour la transformation de plantes
WO1998016824A1 (fr) * 1996-10-15 1998-04-23 Heinz Ganzelmeier Procede de detection optique et de traitement de plantes cultivees et de plantes cultivees transformees non biologiquement
US5777079A (en) * 1994-11-10 1998-07-07 The Regents Of The University Of California Modified green fluorescent proteins
WO1998030701A1 (fr) * 1997-01-14 1998-07-16 Pioneer Hi-Bred International, Inc. Procede d'amelioration de l'efficacite de transformation
US5804387A (en) * 1996-02-01 1998-09-08 The Board Of Trustees Of The Leland Stanford Junior University FACS-optimized mutants of the green fluorescent protein (GFP)
EP0851868A4 (fr) * 1995-09-22 1998-11-25 Gen Hospital Corp Expression de proteines a des hauts niveaux
US5912137A (en) * 1996-07-16 1999-06-15 The Regents Of The University Of California Assays for protein kinases using fluorescent
US5925558A (en) * 1996-07-16 1999-07-20 The Regents Of The University Of California Assays for protein kinases using fluorescent protein substrates
US5981200A (en) * 1996-01-31 1999-11-09 The Regents Of The University Of California Tandem fluorescent protein constructs
US5989835A (en) * 1997-02-27 1999-11-23 Cellomics, Inc. System for cell-based screening
US5998204A (en) * 1997-03-14 1999-12-07 The Regents Of The University Of California Fluorescent protein sensors for detection of analytes
WO2000008054A1 (fr) * 1998-08-08 2000-02-17 Imperial Cancer Research Technology Limited Proteine a fluorescence verte modifiee
US6046925A (en) * 1997-04-14 2000-04-04 The Regents Of The University Of California Photochromic fluorescent proteins and optical memory storage devices based on fluorescent proteins
US6054321A (en) * 1996-08-16 2000-04-25 The Regents Of The University Of California Long wavelength engineered fluorescent proteins
US6066476A (en) * 1994-11-10 2000-05-23 The Regents Of The University Of California Modified green fluorescent proteins
US6090919A (en) * 1997-01-31 2000-07-18 The Board Of Trustees Of The Leland Stanford Junior University FACS-optimized mutants of the green fluorescent protein (GFP)
US6114148A (en) * 1996-09-20 2000-09-05 The General Hospital Corporation High level expression of proteins
US6159705A (en) * 1996-09-24 2000-12-12 Cadus Pharmaceutical Corporation Recombinant yeast cells for identifying receptor effectors
NL1012782C2 (nl) * 1999-08-05 2001-02-06 Keygene Nv Verbeterde gfp-variant, bruikbaar als markering, en gfp-construct.
US6194548B1 (en) 1998-01-23 2001-02-27 Takashi Osumi Green fluorescent proteins and blue fluorescent proteins
US6197928B1 (en) 1997-03-14 2001-03-06 The Regents Of The University Of California Fluorescent protein sensors for detection of analytes
US6255558B1 (en) 1996-02-14 2001-07-03 Btg International Limited Gene expression
WO2001004331A3 (fr) * 1999-07-10 2001-07-19 Calgene Llc Amelioration de l'expression de proteines
US6410255B1 (en) 1999-05-05 2002-06-25 Aurora Biosciences Corporation Optical probes and assays
US6495664B1 (en) 1998-07-24 2002-12-17 Aurora Biosciences Corporation Fluorescent protein sensors of post-translational modifications
WO2003029286A1 (fr) * 2001-09-28 2003-04-10 Amersham Biosciences Uk Limited Proteines fluorescentes
US6593135B2 (en) 1996-08-16 2003-07-15 The State Of Oregon, Acting By And Through The State Board Of Higher Education On Behalf Of The University Of Oregon Long wavelength engineered fluorescent proteins
US6671624B1 (en) 1997-02-27 2003-12-30 Cellomics, Inc. Machine readable storage media for detecting distribution of macromolecules between nucleus and cytoplasm in cells
US6716588B2 (en) 1999-12-09 2004-04-06 Cellomics, Inc. System for cell-based screening
US6727071B1 (en) 1997-02-27 2004-04-27 Cellomics, Inc. System for cell-based screening
EP1441032A1 (fr) 1997-06-05 2004-07-28 Duke University Procédés d'analyse de l'activité de récepteurs et produits de recombinaison utiles dans de tels procédés
US6900304B2 (en) 1996-01-31 2005-05-31 The Regents Of The University Of California Emission ratiometric indicators of phosphorylation
US6919186B2 (en) 2001-04-23 2005-07-19 Amersham Plc Fluorescent proteins
US6986993B1 (en) 1999-08-05 2006-01-17 Cellomics, Inc. System for cell-based screening
US7109315B2 (en) 2000-03-15 2006-09-19 Bruce J. Bryan Renilla reniformis fluorescent proteins, nucleic acids encoding the fluorescent proteins and the use thereof in diagnostics, high throughput screening and novelty items
US7117098B1 (en) 1997-02-27 2006-10-03 Cellomics, Inc. Machine-readable storage medium for analyzing distribution of macromolecules between the cell membrane and the cell cytoplasm
EP1925320A2 (fr) 1998-03-27 2008-05-28 Prolume, Ltd. Luciférases, protéines fluorescentes, acides nucléiques codant pour les luciférases et protéines fluorescentes et leur utilisation pour le diagnostic, le criblage à haut rendement et articles nouveaux
WO2009020197A1 (fr) 2007-08-03 2009-02-12 National University Corporation Hokkaido University Protéine fluorescente avec couleur bleue nuit
US7541151B2 (en) 1997-06-05 2009-06-02 Duke University Single-cell biosensor for the measurement of GPCR ligands in a test sample
US7572888B2 (en) 1997-06-05 2009-08-11 Duke University Methods of assaying receptor activity and constructs useful in such methods
US7582461B2 (en) 2003-07-29 2009-09-01 Life Technologies Corporation Kinase and phosphatase assays
US7595375B2 (en) 2001-06-08 2009-09-29 Riken Fluorescent protein
US7619059B2 (en) 2003-07-29 2009-11-17 Life Technologies Corporation Bimolecular optical probes
EP2221363A1 (fr) 2000-12-27 2010-08-25 Axiogenesis Ag Système pour la sélection spécifique au type de cellule et au stade de développement de celulles souche embryonnaires non-humaines, de cellules germinales embryonnaires non-humaines, et de celulles souche adultes pendant un processus de différentiation
EP2270196A2 (fr) 2004-05-11 2011-01-05 Axiogenesis Ag Methodes pour la découverte de médicaments utilisant des cellules différentiées in vitro
EP2325308A2 (fr) 1997-04-04 2011-05-25 The Board of Regents of the University of Nebraska Procédés et matériaux pour la fabrication et l'utilisation d'organismes transgéniques dégradant le dicamba
WO2012098260A1 (fr) 2011-01-21 2012-07-26 Axiogenesis Ag Système non viral pour générer des cellules souches pluripotentes induites (ips)
EP2482075A1 (fr) 2004-12-01 2012-08-01 Proteologics, Ltd Analyses de l'ubiquitine ligase et réactifs correspondants
EP2578676A1 (fr) 2003-06-20 2013-04-10 Axiogenesis Ag Formation de tissus dans un système de cellules souches embryonnaires (ES)
US20150203793A1 (en) * 2012-08-22 2015-07-23 Novozymes A/S Metalloprotease from Exiguobacterium
WO2018015389A1 (fr) 2016-07-18 2018-01-25 University Of Bremen Procédé de génération de plantes polyploïdes
US9945840B2 (en) 2004-04-07 2018-04-17 Axiogenesis Ag Non-invasive, in vitro functional tissue assay systems
WO2021035325A1 (fr) 2019-08-27 2021-03-04 Fundação Oswaldo Cruz Réceptacle protéique, polynucléotide, vecteur, cassette d'expression, cellule, procédé de production du réceptacle, procédé d'identification de pathogènes ou de diagnostic de maladies, utilisation du réceptacle et d'une trousse de diagnostic

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1991001305A1 (fr) * 1989-07-22 1991-02-07 University Of Wales College Of Medicine Proteines bioluminescentes modifiees et leur utilisation
WO1995007463A1 (fr) * 1993-09-10 1995-03-16 The Trustees Of Columbia University In The City Of New York Methodes d'utilisation de proteines fluorescentes vertes

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1991001305A1 (fr) * 1989-07-22 1991-02-07 University Of Wales College Of Medicine Proteines bioluminescentes modifiees et leur utilisation
WO1995007463A1 (fr) * 1993-09-10 1995-03-16 The Trustees Of Columbia University In The City Of New York Methodes d'utilisation de proteines fluorescentes vertes

Non-Patent Citations (6)

* Cited by examiner, † Cited by third party
Title
CHLAFIE, M., ET AL.: "Green fluorescent protein as a marker for gene expression", SCIENCE, vol. 263, 11 February 1994 (1994-02-11), pages 802 - 805, XP002003599 *
CHUI, W., ET AL.: "Engineered GFP as a vital reporter plants", CURR. BIOL. (1996), 6(3), 325, pages 325 - 330, XP000571865 *
HASELOFF, J., ET AL.: "GFP in plants", TRENDS IN GENETICS, vol. 11, no. 8, August 1995 (1995-08-01), pages 328 - 329, XP002003595 *
NIEDZ, R.P., ET AL.: "Green fluorescent protein : an in vivo reporter of plant gene expression", PLANT CELL REPORTS, vol. 14, April 1995 (1995-04-01), pages 403 - 406, XP000571886 *
SHEEN, J., ET AL.: "Green-fluorescent protein as a new vital marker in plant cells", THE PLANT JOURNAL, vol. 8, no. 5, November 1995 (1995-11-01), pages 777 - 784, XP002003596 *
WANG, S., ET AL.: "Implications for bcd mRNA localization from spatial ditribution of exu protein in Drosophila oogenesis", NATURE, vol. 369, June 1994 (1994-06-01), pages 400 - 403, XP002003600 *

Cited By (86)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6066476A (en) * 1994-11-10 2000-05-23 The Regents Of The University Of California Modified green fluorescent proteins
US6800733B2 (en) 1994-11-10 2004-10-05 The Regents Of The University Of California Modified green fluorescent proteins
US6319669B1 (en) 1994-11-10 2001-11-20 The Regents Of The University Of California Modified green fluorescent proteins
US5777079A (en) * 1994-11-10 1998-07-07 The Regents Of The University Of California Modified green fluorescent proteins
EP0851868A4 (fr) * 1995-09-22 1998-11-25 Gen Hospital Corp Expression de proteines a des hauts niveaux
US8679742B2 (en) 1996-01-31 2014-03-25 The Regents Of The University Of California Method for determining the presence of an enzyme in a sample
US5981200A (en) * 1996-01-31 1999-11-09 The Regents Of The University Of California Tandem fluorescent protein constructs
US6803188B1 (en) 1996-01-31 2004-10-12 The Regents Of The University Of California Tandem fluorescent protein constructs
US6900304B2 (en) 1996-01-31 2005-05-31 The Regents Of The University Of California Emission ratiometric indicators of phosphorylation
US5804387A (en) * 1996-02-01 1998-09-08 The Board Of Trustees Of The Leland Stanford Junior University FACS-optimized mutants of the green fluorescent protein (GFP)
US6255558B1 (en) 1996-02-14 2001-07-03 Btg International Limited Gene expression
WO1997040178A1 (fr) * 1996-04-19 1997-10-30 Mycogen Plant Science, Inc. SURVEILLANCE DE PLANTES TRANSGENIQUES A MARQUEURS $i(IN VIVO)
US6486382B1 (en) 1996-05-01 2002-11-26 Pioneer Hi-Bred International, Inc. Use of the green fluorescent protein as a screenable marker for plant transformation
WO1997041228A3 (fr) * 1996-05-01 1997-12-11 Pioneer Hi Bred Int Utilisation de la proteine fluorescente verte comme marqueur de criblage pour la transformation de plantes
US5912137A (en) * 1996-07-16 1999-06-15 The Regents Of The University Of California Assays for protein kinases using fluorescent
US5925558A (en) * 1996-07-16 1999-07-20 The Regents Of The University Of California Assays for protein kinases using fluorescent protein substrates
US6248550B1 (en) 1996-07-16 2001-06-19 The Regents Of The University Of California Assays for protein kinases using fluorescent protein substrates
US7560287B2 (en) 1996-08-16 2009-07-14 The Regents Of The University Of California Long wavelength engineered fluorescent proteins
US6054321A (en) * 1996-08-16 2000-04-25 The Regents Of The University Of California Long wavelength engineered fluorescent proteins
US8263412B2 (en) 1996-08-16 2012-09-11 The State Of Oregon, Acting By And Through The State Board Of Higher Education On Behalf Of The University Of Oregon Long wavelength engineered fluorescent proteins
US6780975B2 (en) 1996-08-16 2004-08-24 The Regents Of The University Of California Long wavelength engineered fluorescent proteins
US6593135B2 (en) 1996-08-16 2003-07-15 The State Of Oregon, Acting By And Through The State Board Of Higher Education On Behalf Of The University Of Oregon Long wavelength engineered fluorescent proteins
US7544776B2 (en) 1996-08-16 2009-06-09 The University Of Oregon Long wavelength engineered fluorescent proteins
US6077707A (en) * 1996-08-16 2000-06-20 The Regents Of The University Of California Long wavelength engineered fluorescent proteins
US6403374B1 (en) 1996-08-16 2002-06-11 The Regents Of The University Of California Long wavelength engineered fluorescent proteins
US6114148A (en) * 1996-09-20 2000-09-05 The General Hospital Corporation High level expression of proteins
US6159705A (en) * 1996-09-24 2000-12-12 Cadus Pharmaceutical Corporation Recombinant yeast cells for identifying receptor effectors
WO1998016824A1 (fr) * 1996-10-15 1998-04-23 Heinz Ganzelmeier Procede de detection optique et de traitement de plantes cultivees et de plantes cultivees transformees non biologiquement
AU729122B2 (en) * 1997-01-14 2001-01-25 Pioneer Hi-Bred International, Inc. Methods for improving transformation efficiency
US6096947A (en) * 1997-01-14 2000-08-01 Pioneer Hi-Bred International, Inc. Methods for improving transformation efficiency
WO1998030701A1 (fr) * 1997-01-14 1998-07-16 Pioneer Hi-Bred International, Inc. Procede d'amelioration de l'efficacite de transformation
US6090919A (en) * 1997-01-31 2000-07-18 The Board Of Trustees Of The Leland Stanford Junior University FACS-optimized mutants of the green fluorescent protein (GFP)
US7060445B1 (en) 1997-02-27 2006-06-13 Cellomics, Inc. System for cell-based screening
US7235373B2 (en) 1997-02-27 2007-06-26 Cellomics, Inc. System for cell-based screening
US6573039B1 (en) 1997-02-27 2003-06-03 Cellomics, Inc. System for cell-based screening
US5989835A (en) * 1997-02-27 1999-11-23 Cellomics, Inc. System for cell-based screening
US6620591B1 (en) 1997-02-27 2003-09-16 Cellomics, Inc. System for cell-based screening
US6671624B1 (en) 1997-02-27 2003-12-30 Cellomics, Inc. Machine readable storage media for detecting distribution of macromolecules between nucleus and cytoplasm in cells
US6902883B2 (en) 1997-02-27 2005-06-07 R. Terry Dunlay System for cell-based screening
US6727071B1 (en) 1997-02-27 2004-04-27 Cellomics, Inc. System for cell-based screening
US7117098B1 (en) 1997-02-27 2006-10-03 Cellomics, Inc. Machine-readable storage medium for analyzing distribution of macromolecules between the cell membrane and the cell cytoplasm
US7853409B2 (en) 1997-02-27 2010-12-14 Cellomics, Inc. System for cell-based screening
US5998204A (en) * 1997-03-14 1999-12-07 The Regents Of The University Of California Fluorescent protein sensors for detection of analytes
US7993879B2 (en) 1997-03-14 2011-08-09 The Regents Of The University Of California Fluorescent protein sensors for detection of analytes
US6197928B1 (en) 1997-03-14 2001-03-06 The Regents Of The University Of California Fluorescent protein sensors for detection of analytes
EP2325308A2 (fr) 1997-04-04 2011-05-25 The Board of Regents of the University of Nebraska Procédés et matériaux pour la fabrication et l'utilisation d'organismes transgéniques dégradant le dicamba
US6046925A (en) * 1997-04-14 2000-04-04 The Regents Of The University Of California Photochromic fluorescent proteins and optical memory storage devices based on fluorescent proteins
EP1441032A1 (fr) 1997-06-05 2004-07-28 Duke University Procédés d'analyse de l'activité de récepteurs et produits de recombinaison utiles dans de tels procédés
US7572888B2 (en) 1997-06-05 2009-08-11 Duke University Methods of assaying receptor activity and constructs useful in such methods
US7541151B2 (en) 1997-06-05 2009-06-02 Duke University Single-cell biosensor for the measurement of GPCR ligands in a test sample
US7138240B2 (en) 1997-06-05 2006-11-21 Duke University Methods of assaying receptor activity
US7198916B2 (en) 1998-01-23 2007-04-03 Takashi Osumi Green fluorescent proteins and blue fluorescent proteins
US6194548B1 (en) 1998-01-23 2001-02-27 Takashi Osumi Green fluorescent proteins and blue fluorescent proteins
EP1925320A2 (fr) 1998-03-27 2008-05-28 Prolume, Ltd. Luciférases, protéines fluorescentes, acides nucléiques codant pour les luciférases et protéines fluorescentes et leur utilisation pour le diagnostic, le criblage à haut rendement et articles nouveaux
US6495664B1 (en) 1998-07-24 2002-12-17 Aurora Biosciences Corporation Fluorescent protein sensors of post-translational modifications
WO2000008054A1 (fr) * 1998-08-08 2000-02-17 Imperial Cancer Research Technology Limited Proteine a fluorescence verte modifiee
US6410255B1 (en) 1999-05-05 2002-06-25 Aurora Biosciences Corporation Optical probes and assays
WO2001004331A3 (fr) * 1999-07-10 2001-07-19 Calgene Llc Amelioration de l'expression de proteines
US8062856B2 (en) 1999-08-05 2011-11-22 Cellomics, Inc. System for cell-based screening
NL1012782C2 (nl) * 1999-08-05 2001-02-06 Keygene Nv Verbeterde gfp-variant, bruikbaar als markering, en gfp-construct.
US6986993B1 (en) 1999-08-05 2006-01-17 Cellomics, Inc. System for cell-based screening
US6716588B2 (en) 1999-12-09 2004-04-06 Cellomics, Inc. System for cell-based screening
US7109315B2 (en) 2000-03-15 2006-09-19 Bruce J. Bryan Renilla reniformis fluorescent proteins, nucleic acids encoding the fluorescent proteins and the use thereof in diagnostics, high throughput screening and novelty items
EP2221363A1 (fr) 2000-12-27 2010-08-25 Axiogenesis Ag Système pour la sélection spécifique au type de cellule et au stade de développement de celulles souche embryonnaires non-humaines, de cellules germinales embryonnaires non-humaines, et de celulles souche adultes pendant un processus de différentiation
US6919186B2 (en) 2001-04-23 2005-07-19 Amersham Plc Fluorescent proteins
US7091317B2 (en) 2001-04-23 2006-08-15 Ge Healthcare Limited Fluorescent proteins
US7300762B2 (en) 2001-04-23 2007-11-27 Ge Healthcare Uk Limited Fluorescent proteins and methods of using same
US7595375B2 (en) 2001-06-08 2009-09-29 Riken Fluorescent protein
US7981658B2 (en) 2001-06-08 2011-07-19 Riken Nucleic acid molecules, vectors, and host cells encoding fluorescent protein
WO2003029286A1 (fr) * 2001-09-28 2003-04-10 Amersham Biosciences Uk Limited Proteines fluorescentes
EP2578676A1 (fr) 2003-06-20 2013-04-10 Axiogenesis Ag Formation de tissus dans un système de cellules souches embryonnaires (ES)
EP3184629A1 (fr) 2003-06-20 2017-06-28 Axiogenesis Ag Découverte de médicaments à base de tissu produit in vitro
US7582461B2 (en) 2003-07-29 2009-09-01 Life Technologies Corporation Kinase and phosphatase assays
US7619059B2 (en) 2003-07-29 2009-11-17 Life Technologies Corporation Bimolecular optical probes
US11835433B2 (en) 2004-04-07 2023-12-05 Evotec International Gmbh Non-invasive, in vitro functional tissue assay systems
US9945840B2 (en) 2004-04-07 2018-04-17 Axiogenesis Ag Non-invasive, in vitro functional tissue assay systems
EP3070174A1 (fr) 2004-05-11 2016-09-21 Axiogenesis Ag Découverte de médicaments utilisant des cellules différentiées in vitro
EP2270196A2 (fr) 2004-05-11 2011-01-05 Axiogenesis Ag Methodes pour la découverte de médicaments utilisant des cellules différentiées in vitro
US9726662B2 (en) 2004-05-11 2017-08-08 Axiogenesis Ag Assay for drug discovery based on in vitro differentiated cells
US8318488B1 (en) 2004-05-11 2012-11-27 Axiogenesis Ag Assay for drug discovery based on in vitro differentiated cells
EP2482075A1 (fr) 2004-12-01 2012-08-01 Proteologics, Ltd Analyses de l'ubiquitine ligase et réactifs correspondants
WO2009020197A1 (fr) 2007-08-03 2009-02-12 National University Corporation Hokkaido University Protéine fluorescente avec couleur bleue nuit
WO2012098260A1 (fr) 2011-01-21 2012-07-26 Axiogenesis Ag Système non viral pour générer des cellules souches pluripotentes induites (ips)
US20150203793A1 (en) * 2012-08-22 2015-07-23 Novozymes A/S Metalloprotease from Exiguobacterium
WO2018015389A1 (fr) 2016-07-18 2018-01-25 University Of Bremen Procédé de génération de plantes polyploïdes
WO2021035325A1 (fr) 2019-08-27 2021-03-04 Fundação Oswaldo Cruz Réceptacle protéique, polynucléotide, vecteur, cassette d'expression, cellule, procédé de production du réceptacle, procédé d'identification de pathogènes ou de diagnostic de maladies, utilisation du réceptacle et d'une trousse de diagnostic

Also Published As

Publication number Publication date
GB9504446D0 (en) 1995-04-26
AU4884396A (en) 1996-09-23

Similar Documents

Publication Publication Date Title
WO1996027675A1 (fr) Expression de la proteine fluorescente verte (pfv) de meduse dans des plantes
AU730927B2 (en) Use of the green fluorescent protein as a screenable marker for plant transformation
CA2205006C (fr) Proteines fluorescentes vertes modifiees
Reichel et al. Enhanced green fluorescence by the expression of an Aequorea victoria green fluorescent protein mutant in mono-and dicotyledonous plant cells.
AU730040B2 (en) Renilla luciferase and green fluorescent protein fusion genes
Grebenok et al. Green‐fluorescent protein fusions for efficient characterization of nuclear targeting
Mano et al. An Arabidopsis dynamin‐related protein, DRP3A, controls both peroxisomal and mitochondrial division
EP0874903B1 (fr) Genes humanises de la proteine vert fluorescent et procedes
WO1997041228A9 (fr) Utilisation de la proteine fluorescente verte comme marqueur de criblage pour la transformation de plantes
WO1999003997A1 (fr) Genes humanises de proteines fluorescentes vertes et procede d'utilisation associes
HU212920B (en) Process for the preparation of human superoxide dismotase
SK111693A3 (en) Male-sterile plants, methods for obtaining male-sterile plants and recombinant dna for use therein
US6232099B1 (en) Method of producing a chimeric protein
BG61275B1 (en) Production of plants resistant to attacks of sclerotiniasp.by introducing a gene coding for an oxalate oxidase
US6548642B1 (en) Synthetic genes for plant gums
CN1329670A (zh) 编码野生型cop1基因及其螺旋结构域的植物和植物细胞
WO1996040882A1 (fr) Endonuclease artificielle de restriction
US6570062B1 (en) Synthetic genes for plant gums and other hydroxyproline-rich glycoproteins
AU1350801A (en) Production of functional hybrid genes and proteins
CA2502657C (fr) Utilisation de la proteine fluorescente verte comme marqueur de criblage pour la transformation de plantes
CA2079027A1 (fr) Expression accrue de polypeptides recombinants a faible poids moleculaire
NL1012782C2 (nl) Verbeterde gfp-variant, bruikbaar als markering, en gfp-construct.
KR102252056B1 (ko) 재조합 벡터 및 이를 이용한 재조립 시토크롬 p450 옥시게나아제-리덕타아제 융합 단백질의 제조 방법
CN107188939A (zh) Psa3蛋白在参与psi的组装以及维持psi稳定中的应用
Haseloff Imaging green fluorescent protein in transgenic plants

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): AU CA JP US

AL Designated countries for regional patents

Kind code of ref document: A1

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

DFPE Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101)
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