WO2013087921A1 - Protéines fluorescentes génétiquement modifiées à des fins d'amélioration du fret et leurs utilisations - Google Patents

Protéines fluorescentes génétiquement modifiées à des fins d'amélioration du fret et leurs utilisations Download PDF

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WO2013087921A1
WO2013087921A1 PCT/EP2012/075737 EP2012075737W WO2013087921A1 WO 2013087921 A1 WO2013087921 A1 WO 2013087921A1 EP 2012075737 W EP2012075737 W EP 2012075737W WO 2013087921 A1 WO2013087921 A1 WO 2013087921A1
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protein
donor
pair
acceptor
polypeptide
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Raik GRÜNBERG
Luis Serrano Pubul
François STRICHER
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Fundació Privada Centre De Regulació Genòmica (Crg)
Institució Catalana De Recerca I Estudis Avançats (Icrea)
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Publication of WO2013087921A1 publication Critical patent/WO2013087921A1/fr

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/536—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase
    • G01N33/542—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase with steric inhibition or signal modification, e.g. fluorescent quenching
    • 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

Definitions

  • the invention relates to the field of fluorescent proteins and their versatile applications in the field of molecular biology. More specifically, the invention relates to pairs of genetically engineered fluorescent proteins and their uses in FRET-based applications, as well as methods to produce genetically engineered fluorescent proteins for high efficiency FRET (Forster Resonance Energy Transfer) based on the introduction of electrostatic helper interactions.
  • FRET Form Resonance Energy Transfer
  • Fluorescent proteins are widely known today for their use as fluorescent markers in biomedical sciences. They are applied for a wide range of applications including the study of gene expression, protein localization, visualizing subcellular organelles in cells, visualizing protein localization and transport, as well as for detecting protein-protein interactions, or for screening purposes, amongst many others. Due to the potential for widespread usage and the evolving needs of researchers, novel fluorescent proteins have been identified with improved fluorescence intensity and maturation rates at physiological temperatures, modified excitation and emission spectra, and reduced oligomerization and aggregation properties. In addition, mutagenesis of known proteins has been undertaken to improve their chemical properties. Finally, codon usage has been optimized for high expression in the desired heterological system, for example in mammalian cells.
  • FRET Form Resonance Energy Transfer
  • ECFP and EYFP are both derived from Aequorea GFP with a tendency to homodimerize (K D ⁇ 0.1 M) [5].
  • the large improvement in FRET was later shown to be caused mainly by only two mutations that re-enforce the native homodimerization interface stemming from Aequorea GFP [6, 7] - leading to the formation of a high FRET intramolecular complex between CyPet and YPet.
  • Such direct interactions among fluorescent proteins are traditionally considered annoyance [5] rather than virtue.
  • GFP-derived donor and acceptor domains can be rationally enhanced or weakened through the introduction of point mutations among the residues that mediate the intermolecular contact [6,7 and published patent application WO 97/28261]. These residues can be inferred from the crystal structure of the GFP homodimer.
  • Enhanced dimerization can be achieved through the introduction of additional hydrophobic contacts or the introduction of complementary electrostatic charges or both. Electrostatic charges can also be added indirectly, by engineering metal ion binding sites within the dimerization interface [40].
  • all these approaches have several disadvantages. First, as they rely on improving the native GFP homodimerization interface, these methods are limited to pairs of fluorescent proteins where both donor and acceptor are derived from Aequorea GFP.
  • the introduction of molecular contacts within the interface stabilizes the formation of an actual donor/acceptor complex with reduced off rate or, correspondingly, a longer life time and tighter binding.
  • Increased affinities can lead to undesired background signal of a FRET sensor which may eliminate any advantage of overall FRET increase [9].
  • Increased binding and life times may also affect the switching dynamics and reversibility of FRET sensors.
  • helper interactions weak but strictly heterodimeric interactions
  • helper interaction FRET hiFRET
  • One of these methods is based on computational protein design and allowed us to create interfaces for electrostatically driven encounter complexes between unrelated fluorescent proteins.
  • Our helper interaction(s) led to a large increase of FRET efficiency while background signals in the unbound state remained near zero.
  • the enhanced red-shifted FRET pairs will be of immediate use for in vitro and in vivo FRET-based applications. They provide an attractive alternative to classic CFP/YFP pairs.
  • the invention relates to a method of producing a pair of engineered fluorescent proteins having an increased FRET efficiency relative to the non-engineered pair comprising the steps of: a. Providing a pair of fluorescent proteins suitable as a donor and acceptor for FRET measurements,
  • step b in the above described method is further characterized in that it comprises the steps of: a. Creating an in silico structural model of a heterodimeric complex of said pair of fluorescent proteins,
  • the methods according to the invention as described hereinbefore may further comprise the steps of introducing mutations that remove unfavorable contacts within the contact interface of said model heterodimeric complex, and/or introducing mutations that disrupt homodimer interfaces without impairing heterodimerisation.
  • the step of introducing complementary electrostatic charges in any of the above described methods may be further defined as introducing at least one amino acid substitution in at least one of the fluorescent proteins of the pair, preferably at least one positive charge in one fluorescent protein and at least one negative charge in the other fluorescent protein.
  • the fluorescent proteins of the pair suitable as a donor and acceptor for FRET measurements to be provided in any of the above described methods do not show initial intrinsic interaction.
  • said pair of fluorescent proteins may comprise (m)Citrine (SEQ ID NO: 1), or a variant thereof, and mCherry (SEQ ID NO: 4), or a variant thereof.
  • said pair of engineered fluorescent proteins may comprise: a. a donor fluorescent protein comprising at least one amino acid substitution at a position corresponding to a position in SEQ ID NO: 1, selected from the group consisting of positions 43, 144, 202, 221 and 227, and
  • an acceptor fluorescent protein comprising at least one amino acid substitution at a position corresponding to a position in SEQ ID NO: 4, selected from the group consisting of positions 39, 144, 145 and 196.
  • the at least one amino acid substitution may be selected from the group consisting of T43E, T43D, N144E, N144D, S202E, S202D, L221A, A227E and A227D in SEQ ID NO: 1, and from the group consisting of E39K, E39R, E144K, E144R, A145K, A145R, N196K and N196R in SEQ ID NO: 4.
  • Another aspect of the present invention relates to a pair of engineered fluorescent proteins having an increased FRET efficiency relative to a pair of SEQ ID NO: 1 and SEQ ID NO: 4, comprising a donor and an acceptor fluorescent protein, wherein said donor and acceptor fluorescent proteins may be chosen from the group consisting of: a. a donor fluorescent protein comprising at least one amino acid substitution at a position corresponding to a position in SEQ ID NO: 1 selected from the group consisting of positions 43, 144, 202, 221 and 227, and an acceptor fluorescent protein corresponding to SEQ ID NO: 4;
  • a donor fluorescent protein corresponding to SEQ ID NO: 1 a donor fluorescent protein corresponding to SEQ ID NO: 1, and an acceptor fluorescent protein comprising at least one amino acid substitution at a position corresponding to a position in SEQ ID NO: 4 selected from the group consisting of positions 39, 144, 145 and 196.
  • a donor fluorescent protein comprising at least one amino acid substitution at a position corresponding to a position in SEQ ID NO: 1 selected from the group consisting of positions 43, 144, 202, 221 and 227, and an acceptor fluorescent protein comprising at least one amino acid substitution at a position corresponding to a position in SEQ ID NO: 4 selected from the group consisting of positions 39, 144, 145 and 196;
  • the at least one amino acid substitution in the above described pair of engineered fluorescent proteins may be selected from the group consisting of T43E, T43D, N144E, N144D, S202E, S202D, L221A, A227E and A227D in SEQ ID NO: 1, and from the group consisting of E39 , E144K, E144R, A145K, A145R, N196K and N196R in SEQ ID NO: 4.
  • a pair of engineered fluorescent proteins wherein the donor and the acceptor fluorescent proteins of the present invention are each fused to a polypeptide of interest, optionally through a linker molecule, is also provided. Accordingly, bimolecular and unimolecular constructs are also encompassed in the present invention.
  • the invention provides a bimolecular construct, comprising: a. a donor fluorescent protein of the invention fused to a polypeptide of interest, optionally through one or more linker molecules, and
  • an acceptor fluorescent protein of the invention fused to a polypeptide of interest, optionally through one or more linker molecules.
  • the invention provides a unimolecular protein construct selected from the group comprising the following fusion proteins: a. a donor fluorescent protein - a polypeptide of interest - an acceptor fluorescent protein, optionally fused through one or more linker molecules, or
  • a polypeptide of interest - a donor fluorescent protein - an acceptor fluorescent protein - a polypeptide of interest, optionally fused through one or more linker molecules, or
  • a polypeptide of interest an acceptor fluorescent protein - a donor fluorescent protein - a polypeptide of interest, optionally fused through one or more linker molecules, or
  • Alternative embodiments of the invention encompass (1) an engineered fluorescent protein comprising at least one amino acid substitution at a position corresponding to a position in SEQ ID NO: 1 selected from the group consisting of positions 43, 144, 202, 221 and 227, such as T43E, T43D, N144E, N144D, S202E, S202D, L221A, A227E and A227D; (2) an engineered fluorescent protein comprising at least one amino acid substitution at a position corresponding to a position in SEQ ID NO: 4 selected from the group consisting of positions 39, 144 and 145, such as E39 , E144K, E144R, A145K, A145R, N196K and N196R; (3) a fusion protein comprising an engineered fluorescent protein as defined in (1) or (2) fused to a polypeptide of interest, optionally through one or more linker molecules.
  • an engineered fluorescent protein comprising at least one amino acid substitution at a position corresponding to a position in SEQ ID NO: 1 selected from the group
  • polynucleotides encoding any of the donor and/or acceptor fluorescent proteins, or any of the engineered fluorescent proteins, or any of the bimolecular/unimolecular constructs, or any of the fusion proteins, according to the present invention are also envisaged here, as well as expression vectors comprising suitable expression control sequences operably linked to any of the above described polynucleotides, as well as host cells comprising any of the polynucleotides or any of the expression vectors of the invention, as well as antibodies that specifically bind to the engineered fluorescent proteins.
  • the invention provides a kit comprising any of the polynucleotides or any of the expression vectors of the invention as described hereinbefore.
  • Still another aspect of the invention relates to the use of the pair of engineered fluorescent proteins, or the protein constructs, or the individual engineered fluorescent proteins, or the fusion proteins, or any of the polynucleotides or any of the expression vectors, all as defined hereinbefore, for in vitro and/or in vivo FRET-based applications.
  • a method of identifying a specific interaction of a first polypeptide of interest and a second polypeptide of interest comprising: a. Contacting the first polypeptide of interest, which is fused to a donor fluorescent protein of a pair of engineered fluorescent proteins according to the invention, and the second polypeptide of interest, which is fused to the corresponding acceptor fluorescent protein of the pair of engineered fluorescent proteins according to the invention, under conditions that allow a specific interaction of the first and the second polypeptide of interest,
  • Figure l Principle of enhanced FRET.
  • (b, c) Schematic description of protein interaction detection by FRET between conventional (b) and enhanced (c) FRET probes. The drug-induced interaction between FRB and FKBP12 (gray) is used to quantify FRET signals in unbound and bound state.
  • Figure 2 Design of FRET helper interactions.
  • Electrostatically enhanced FRET probes yield higher FRET efficiencies than conventional probes (gray) and are less affected by prolongation of the linker with the FRB/FKBP12 reference interaction.
  • FRET pairs Top: The best performing FRET pairs were mixed at 1:1 stoichiometry (0.5 ⁇ , 24aa linker between binding domain and FP) and their interaction induced with rapamycin. The performance of the conventional FRET pair is shown in gray (unmodified Citrine / mCherry). Quenching of the donor emission by FRET is apparent for all pairs and is more pronounced for constructs with FRET helper interactions. Note, owing to errors in protein concentrations, exact 1:1 ratios are difficult to attain.
  • FIG. 5 Specificity and background activity of helper interactions.
  • the FRET probe pairs with 24 aa linker were tested at high protein concentrations (3 ⁇ [donor] + 5 ⁇ [acceptor]). Gains of FRET efficiency are preserved,
  • Figure 8 Comparison of the original ("wild type") and engineered Citrine sequences. Blue arrows mark positions subject to mutations for the electrostatic interface. Unstructured peptide sequences introduced during the engineering of the original Citrine are labelled as 'spacer'. Additional (wt and 4-) variants with the homodimer-breaking mutation A206K (shown in red without arrow) are omitted for clarity.
  • determining As used herein, the terms “determining,” “measuring,” “assessing,” “testing”, and “assaying” are used interchangeably and include both quantitative and qualitative determinations.
  • polypeptide As used herein, the terms “polypeptide”, “protein”, “peptide”, “oligopeptide” are used interchangeably herein, and refer to a polymeric form of amino acids of any length, which can include coded and non- coded amino acids, chemically or biochemically modified or derivatized amino acids, and polypeptides having modified peptide backbones.
  • nucleic acid molecule As used herein, the terms “nucleic acid molecule”, “polynucleotide”, “polynucleic acid”, “nucleic acid” are used interchangeably and refer to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides, or analogs thereof. Polynucleotides may have any three- dimensional structure, and may perform any function, known or unknown.
  • Non-limiting examples of polynucleotides include a gene, a gene fragment, exons, introns, messenger NA (mRNA), transfer RNA, ribosomal RNA, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, control regions, isolated NA of any sequence, nucleic acid probes, and primers.
  • the nucleic acid molecule may be linear or circular.
  • vector as used herein is intended to refer to a nucleic acid molecule capable of transporting another nucleic acid molecule to which it has been linked.
  • plasmid vector refers to a circular double stranded DNA loop into which additional DNA segments may be ligated.
  • Other vectors include, without the purpose of being limitative, cosmids and yeast artificial chromosomes (YAC).
  • YAC yeast artificial chromosomes
  • Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., vectors having an origin of replication which functions in the host cell).
  • Other vectors can be integrated into the genome of a host cell upon introduction into the host cell, and are thereby replicated along with the host genome.
  • certain preferred vectors are capable of directing the expression of certain genes of interest.
  • Such vectors are referred to herein as “recombinant expression vectors” (or simply, “expression vectors”).
  • Suitable vectors have regulatory sequences, such as promoters, enhancers, terminator sequences, and the like as desired.
  • operably linked refers to a linkage in which the regulatory sequence is contiguous with the gene of interest to control said gene of interest, as well as regulatory sequences that act in trans or at a distance to control the gene of interest.
  • control sequences refers to polynucleotide sequences which are necessary to affect the expression of coding sequences to which they are operatively linked.
  • Expression control sequences are sequences which control the transcription, post-transcriptional events and translation of nucleic acid sequences. Expression control sequences include appropriate transcription initiation, termination, promoter and enhancer sequences; efficient RNA processing signals such as splicing and polyadenylation signals; sequences that stabilize cytoplasmic mRNA; sequences that enhance translation efficiency (e.g., ribosome binding sites); sequences that enhance protein stability; and when desired, sequences that enhance protein secretion. The nature of such control sequences differs depending upon the host organism.
  • control sequences is intended to include, at a minimum, all components whose presence is essential for expression, and can also include additional components whose presence is advantageous, for example, leader sequences and fusion partner sequences.
  • recombinant host cell ("expression host cell”, “expression host system”, “expression system” or simply “host cell”), as used herein, is intended to refer to a cell into which a recombinant vector has been introduced. It should be understood that such terms are intended to refer not only to the particular subject cell but also to the progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term “host cell” as used herein.
  • a recombinant host cell may be an isolated cell or cell line grown in culture or may be a cell which resides in a living tissue or organism.
  • a “mutant” or “variant” or “derivative” (or equivalent wordings having the same meaning), as used herein, and used in the context of an amino acid or nucleotide sequence, is meant to encompass a subsequent amino acid or nucleotide sequence that has been derived from a previous amino acid or nucleotide sequence, or reference sequence, either naturally or artificially.
  • a variant or mutant protein of wild type Aequorea GFP (the reference in this case) may have one or more amino acid substitutions, additions, or deletions as compared to said Aequorea wild type GFP amino acid sequence.
  • fluorescent protein mutants or variants are provided in Tables 5 and 6 (examples known in the art), as well as in the Detailed Description part hereinafter (as part of the present invention).
  • a “spectral variant” or “spectral mutant”, as used herein, refers to a fluorescent protein to indicate a mutant fluorescent protein that has a different fluorescence characteristic with respect to the corresponding wild type fluorescent protein.
  • CFP, YFP, ECFP, EYFP-V68L/Q69K, and the like are GFP spectral variants.
  • substitution results from the replacement of one or more amino acids or nucleotides by different amino acids or nucleotides, respectively, as compared to an amino acid sequence or nucleotide sequence of a parental or reference polypeptide or nucleic acid.
  • the substitutions are conservative substitutions.
  • the substitutions are non-conservative substitutions.
  • conservative and non-conservative amino acid substitutions are known to those of ordinary skill in the art. It is understood that a protein or a fragment thereof may have conservative amino acid substitutions which have substantially no effect on the protein's activity.
  • a “deletion”, as used herein, is defined as a change in either amino acid or nucleotide sequence in which one or more amino acid or nucleotide residues, respectively, are absent as compared to an amino acid sequence or nucleotide sequence of a parental or reference polypeptide or nucleic acid.
  • a deletion can involve deletion of about 2, about 5, about 10, up to about 20, up to about 30 or up to about 50 or more amino acids.
  • a protein or a fragment thereof may contain more than one deletion.
  • an “insertion” or “addition”, as used herein, is that change in an amino acid or nucleotide sequence which results from the insertion or addition of one or more amino acid or nucleotide residues, respectively, as compared to an amino acid sequence or nucleotide sequence of a parental or reference polypeptide or nucleic acid.
  • “Insertion” or “addition” generally refers to the addition of one or more amino acid residues within an amino acid sequence of a polypeptide, while “addition” can be an insertion or refer to amino acid residues added at an N- or C-terminus, or both termini.
  • an insertion or addition is usually of about 1 , about 3, about 5, about 10, up to about 20, up to about 30 or up to about 50 or more amino acids.
  • a protein or fragment thereof may contain more than one insertion.
  • antibody refers generally to a polypeptide encoded by an immunoglobulin gene, or antigen-binding fragments thereof, which specifically binds and recognizes an antigen, and is known to the person skilled in the art.
  • a conventional immunoglobulin (antibody) structural unit comprises a tetramer. Each tetramer is composed of two identical pairs of polypeptide chains, each pair having one "light” (about 25 kDa) and one "heavy” chain (about 50-70 kDa). The N-terminus of each chain defines a variable region of about 100 to 110 or more amino acids primarily responsible for antigen recognition.
  • VL variable light chain
  • VH variable heavy chain
  • antigen-binding fragments may be antigen-binding antibody fragments that include, but are not limited to, Fab, Fab' and F(ab')2, Fd, single-chain Fvs (scFv), single-chain antibodies, disulfide-linked Fvs (dsFv) and fragments comprising or consisting of either a VL or VH domain, and any combination of those or any other functional portion of an immunoglobulin peptide capable of binding to the target antigen.
  • antibodies is also meant to include heavy chain antibodies, or functional fragments thereof, such as single domain antibodies, for example, nanobodies.
  • polypeptides specifically interact with a protein domain and not, or to a lesser degree, with other (poly)peptides in a mixture of different polypeptides.
  • a specific binding interaction will discriminate between desirable and undesirable polypeptides in a sample, in some embodiments more than about 10 to 100-fold or more (e.g., more than about 1000- or 10,000-fold).
  • affinity refers to the degree to which one particular polypeptide binds to or interacts with another particular polypeptide so as to shift the equilibrium of either polypeptide toward the presence of a complex formed by their binding.
  • affinity refers to the degree to which one particular polypeptide binds to or interacts with another particular polypeptide so as to shift the equilibrium of either polypeptide toward the presence of a complex formed by their binding.
  • a peptide of low affinity will usually not bind to a protein domain, unless one or both of them are present in a high concentration or if their co-recruitment or protein fusion creates a high local concentration.
  • the dissociation constant is commonly used to describe the affinity between two polypeptides, in particular between the peptide and the protein domain. Typically, moderate to strong interactions (moderate to high affinity) imply that the dissociation constant is lower than 10 s M. Strong interactions (high affinity) imply a dissociation constant lower than 10 -6 M. Conversely, weak interactions typically imply that the dissociation constant is larger than 10 s M.
  • the strong distance dependence of FRET hampers the wide-spread application of genetically encoded FRET pairs.
  • the present invention provides a strategy for the rational engineering of weak helper interactions that align donor and acceptor fluorophores, leading to robust FRET signals without elaborate optimization of linker sequences or orientations.
  • a first aspect of the invention relates to a method of producing a pair of engineered fluorescent proteins having an increased FRET efficiency relative to the non-engineered pair comprising the steps of: a Providing a pair of fluorescent proteins suitable as a donor and acceptor in FRET measurements,
  • the invention also relates to a method of producing a pair of engineered fluorescent proteins having an increased FRET efficiency relative to the non-engineered pair comprising the steps of: a. Providing a pair of fluorescent proteins suitable as a donor and acceptor in FRET measurements,
  • fluorescent protein refers to any protein that can fluoresce when excited with an appropriate electromagnetic radiation.
  • a fluorescent protein may exhibit low, medium or intense fluorescence upon irradiation with light of the appropriate excitation wavelength.
  • the fluorescent proteins of the present invention do not include proteins that exhibit fluorescence only from residues that act by themselves as intrinsic fluorophors, i.e., tryptophan, tyrosine and phenylalanine.
  • a fluorescent protein of the invention or for use in a method of the invention is a protein that derives its fluorescence from autocatalytically forming a chromophore.
  • a fluorescent protein can contain amino acid sequences that are naturally occurring or that have been engineered (i.e., variants or mutants or derivatives, as defined herein).
  • a spectral variant of Aequorea GFP can be derived from the naturally occurring GFP by engineering mutations such as amino acid substitutions into the reference GFP protein.
  • ECFP is a spectral variant of GFP that contains substitutions with respect to GFP.
  • fluorescent protein also includes variants of fluorescent proteins that have lost actual fluorescence but can still act as FRET acceptors by virtue of a "dark quenching" or "dark absorbing” chromophore.
  • Non- limiting examples are the REACh variants of YFP [39]. Fluorescent proteins are often classified according to their spectral class. Thus, fluorescent proteins may be green fluorescent proteins which fluoresce green, or red fluorescent proteins which fluoresce red, or yellow fluorescent proteins which fluoresce yellow, or cyan fluorescent proteins which fluoresce cyan, or orange fluorescent proteins which fluoresce orange, etc.
  • green fluorescent protein or "GFP”is used broadly herein to refer to a protein that fluoresces green light, for example, Aequorea GFP. GFPs have been isolated from the jellyfish, Aequorea victoria, the sea pansy, enilla reniformis, and Phialidium gregarium [21, 23].
  • red fluorescent protein is used in the broadest sense and specifically covers the Discosoma RFP (DsRed), and red fluorescent proteins from any other species, such as coral and sea anemone, as well as variants thereof, as long as they retain the ability to fluoresce red light[24]. Furthermore, reference is also made to the various spectral variants and mutants that have amino acid sequences that are substantially identical to a reference fluorescent protein. Non-limiting examples of commonly known reference fluorescent proteins include, but are not limited to, A. Victoria GFP (Genebank Accession Number M62654.1), Discosoma RFP (DsRed), (Genebank Accession Number AF168419), amongst others, see [15, 26]; U.S. Patent No. 5,625,048; International application PCT/US95/14692, now published as PCT WO96/23810, each of which is incorporated herein by reference.
  • Aequorea GFP-related fluorescent proteins include, for example, wild type (native) Aequorea victoria GFP [26], allelic variants thereof, for example, a variant having a Q80R substitution [16]; and spectral variants of GFP such as CFP, YFP, and enhanced and otherwise modified forms thereof (U.S. Pat. Nos. 6,150,176; 6,124,128; 6,077,707; 6,066,476; 5,998,204; and 5,777,079, each of which is incorporated herein by reference), including GFP-related fluorescent proteins having one or more folding mutations, and fragments of the proteins that are fluorescent, for example, an A. victoria GFP from which the two N-terminal amino acid residues have been removed.
  • Discosoma RFP (DsRed)-related fluorescent proteins include, for example, wild type (native) Discosoma RFP, allelic variants thereof, and spectral variants thereof, such as mPlum, tdTomato, mStrawberry, DsRed Monomer, mOrange[ll].
  • a further class of fluorescent proteins considered here are variants that are derived from other fluorescent proteins like GFP or DsRed or Citrine or mCherry by means of circular permutation. Circular permutation is the fusion of N- and C-terminal of the original protein while simultaneously creating a new N- and C-terminal within the original protein sequence, with the aim to change the orientation of the fluorescent protein within larger protein fusion constructs [17, 19].
  • fluorescent proteins are chosen according to the type of application and experiment, essentially based on critical factors such as emission spectra, brightness, photostability, oligomerization, for which guidance is provided in the art, see e.g. [3].
  • the fluorescent proteins as used herein are suitable for Forster resonance energy transfer (FRET), also known as fluorescence energy transfer, or simply, resonance energy transfer (RET), hereinafter further referred to as "FRET".
  • FRET is the nonradiative transfer of excited-state energy from one molecule (the donor) to another nearby molecule (the acceptor) via a long-range dipole-dipole coupling mechanism, and is well described in the art.
  • the fluorescent proteins as used herein are a pair of fluorescent proteins suitable as a donor and acceptor in FRET measurements, for which guidance is provided in the art, see e.g. [1, 3, 27] as well as in the Detailed Description further herein.
  • the ratio of donor and acceptor fluorescence is measured (also referred to as ratiometric FRET).
  • ratiometric FRET ratio of donor and acceptor fluorescence
  • FLIM fluorescence lifetime imaging microscopy
  • FRET efficiency (£), as used herein, is the quantum yield of the energy transfer transition, i.e. the fraction of energy transfer event occurring per donor excitation event and is known to the person skilled in the art.
  • the FRET efficiency depends on many parameters, including the distance between the donor and the acceptor, the spectral overlap of the donor emission spectrum and the acceptor absorption spectrum, the relative orientation of the donor emission dipole moment and the acceptor dipole moment.
  • the efficiency of FRET between the donor and acceptor is at least 10%, at least 20%, at least 30%, at least 40%, more preferably around 50%.
  • the efficiency of FRET is preferably even higher, at least 60%, at least 70%, and even more preferably at least 80%, at least 90%, or higher.
  • the calculated FRET efficiencies can be compared between two pairs of fluorescent proteins, wherein one pair of fluorescent proteins may have an "increased” or “higher” FRET efficiency or may have a “reduced” or “lower” FRET efficiency relative to the other pair.
  • the difference in FRET efficiency is statistically significant.
  • the rate of energy transfer depends upon the extent of spectral overlap between the donor emission and acceptor absorption spectra, the quantum yield of the donor, the relative orientation of the donor and acceptor transition dipole moments, and the distance separating the donor and acceptor molecules.
  • a preferred factor to be considered in choosing the donor and acceptor pair is the efficiency of fluorescence resonance energy transfer between them.
  • the present invention provides guidance on how to improve FRET efficiency. It will be clear that spectral overlap, quantum yield and related spectroscopic properties are a direct consequence of the choice of donor and acceptor protein variant and remain valid for various implementations or applications of the same FRET pair. These were the main criteria that have been used in the past to optimize the efficiency and detectability of FRET between a donor and acceptor molecule.
  • the present invention offers means to optimize FRET probe distance and orientation largely independent of the molecular and experimental context in which the FRET pair is applied. Distance optimized helper interaction FRET pairs can therefore be used for a variety of applications without or with little further customization.
  • a pair of engineered fluorescent proteins or "an engineered pair of fluorescent proteins”, as used herein, refers to a pair of fluorescent proteins that has been genetically engineered or modified, according to any of the above described engineering methods; this is in contrast to a non-engineered fluorescent protein pair that has not been genetically engineered or modified according to any of the above described engineering methods, and that is known in the art. It should be clear that a non-engineered pair of fluorescent proteins as described herein is not necessarily restricted to a pair of wild type or reference fluorescent proteins, but may also be, for example, a homolog, a spectral variant or another mutant derived thereof (as defined herein).
  • the pair of engineered fluorescent proteins is optimized for higher FRET efficiency as compared to the non-engineered pair. It should be clear that the above described engineering method is meant to improve FRET efficiency by optimizing a pair of fluorescent proteins simultaneously (i.e. donor and acceptor protein), and not the fluorescent proteins individually.
  • the above described method of producing a pair of engineered fluorescent proteins having an increased FRET efficiency relative to the non-engineered pair comprises the step of introducing complementary electrostatic charges in said pair of fluorescent proteins resulting in a pair of engineered fluorescent proteins.
  • complementary electrostatic charges refers to electrostatic interactions between proteins and is mainly determined by the charged residues, in particular at the protein surface.
  • "Introducing complementary electrostatic charges” refers to modifying the number of charged residues of the two proteins in opposite direction, for example, by introducing positively charged amino acids in the first protein and by introducing negatively charged amino acids in the second protein.
  • at least one, at least two, at least three, or more amino acid substitutions in at least one of the fluorescent proteins of the pair are introduced.
  • At least four, at least five, or more amino acid substitutions in at least one of the fluorescent proteins of the pair are introduced.
  • at least one, at least two, at least three, or more amino acid substitutions are introduced in the two fluorescent proteins of the pair (thus in both fluorescent proteins).
  • at least four, at least five, or more amino acid substitutions are introduced in the two fluorescent proteins of the pair.
  • the at least one or more amino acid substitution results in the introduction of at least one positive charge in one fluorescent protein of the pair and the introduction of at least one negative charge in the other fluorescent protein of the pair, or vice versa.
  • introduction of a positive charge means introducing a "net positive charge” by introducing one or more positive charge(s) or removing one or more negative charge(s), or combinations thereof resulting in a net positive charge.
  • introduction of a negative charge means introducing a "net negative charge” by introducing one or more negative charge(s) or removing one or more positive charge(s), or combinations thereof resulting in a net negative charge.
  • the above described method of obtaining a pair of engineered fluorescent proteins having an increased FRET efficiency relative to the non-engineered pair comprises the step of creating an in silico structural model of a heterodimeric complex of a pair of fluorescent proteins that is provided and to use this as a template for optimizing said pair for high FRET efficiency.
  • the design or construction of such a hypothetical structural model between the two fluorescent proteins is typically based on available protein structures in the Protein Data Bank (PDB, www.pdb.org; [20]), preferably of at least one of the constituting fluorescent proteins of the pair or a homolog thereof.
  • an existing structure of a complex of two identical (homodimer) or homologous fluorescent proteins is used as a "template” homodimer complex.
  • the terms “creating” or “design” or “construction” or semantic derivatives thereof are used interchangeably herein.
  • structure- and/or sequence- based alignments are used to position donor and acceptor protein structures into the same orientation as the "template” homodimer complex. This is done by making use of computer algorithms known in the art, for example TM-align, which is illustrated in a non-limiting way further herein in the Example section.
  • a homodimeric complex of two fluorescent proteins means a complex formed by association of two identical fluorescent proteins.
  • a homodimeric interface refers to the residues that are participating in the molecular contacts between the two identical fluorescent proteins of a homodimeric complex.
  • a heterodimeric complex of two fluorescent proteins means a complex formed by association of two distinct fluorescent proteins.
  • a heterodimeric interface refers to the residues that are participating in the molecular contacts between the two distinct proteins of a heterodimeric complex.
  • the complementary electrostatic charges are introduced within or, more preferably, in the direct vicinity of the modelled heterodimeric contact interface between the two fluorescent proteins of the pair, hereby promoting the weak interaction between both proteins of the pair.
  • rim of an interface
  • directly vicinity or "rim” of an interface, as used herein, is defined as those residues that have at least one backbone or C p atom within 1.5 nm of a backbone or C p atom from the partner protein but do not belong to the actual interface (as defined above). That means, all atoms of rim residues are further than 0.5 nm away from all atoms of the partner protein.
  • These atomic distances can preferably be derived from the structure or a structural model of the protein- protein interaction complex.
  • related methods e.g. docking
  • the above described method of obtaining a pair of engineered fluorescent proteins having an increased FRET efficiency relative to the non-engineered pair further comprises the step of introducing mutations that remove unfavorable contacts within the contact interface of said model heterodimeric complex, causing, for example, steric hindrances (also referred to as "clashes") between the binding partners.
  • the above method may further comprise the step of introducing mutations that disrupt homodimer interfaces without impairing heterodimerisation, as can be predicted from existing homodimeric structures of one or both of the constituting fluorescent proteins of the pair (acceptonacceptor and/or donondonor), or from the homodimeric structure of homologous proteins derived thereof.
  • the fluorescent proteins of the pair do not show initial intrinsic interaction, such as fluorescent protein variants that have been made monomeric like mCitrine or mCherry (see also Table 5 and 6).
  • the pair of fluorescent proteins of the method as described herein comprises (m)Citrine (SEQ ID NO: 1), or a variant thereof (as defined hereinbefore), and mCherry (SEQ ID NO: 4), or a variant thereof (as defined hereinbefore).
  • mCitrine SEQ ID NO: 1
  • mCherry SEQ ID NO: 4
  • Citrine is derived from and a variant of GFP
  • mCherry SEQ ID NO: 4
  • mCherry SEQ ID NO: 4
  • the pair of fluorescent proteins of the method as described herein comprises: a. a donor fluorescent protein comprising at least one amino acid substitution at a position corresponding to a position in SEQ ID NO: 1, selected from the group consisting of positions 43, 144, 202, 221 and 227, and
  • an acceptor fluorescent protein comprising at least one amino acid substitution at a position corresponding to a position in SEQ ID NO: 4, selected from the group consisting of positions 39, 144, 145 and 196.
  • the at least one amino acid substitution is selected from the group consisting of T43E, T43D, N144E, N144D, S202E, S202D, L221A, A227E and A227D in SEQ ID NO: l,and from the group consisting of E39K, E39R, E144K, E144R, A145K, A145R, N196K and N196R in SEQ ID NO: 4.
  • a second aspect of the invention relates to a pair of engineered fluorescent proteins having an increased FRET efficiency relative to a pair of SEQ ID NO: 1 and SEQ ID NO: 4, comprising a donor and an acceptor fluorescent protein, wherein said donor and acceptor fluorescent proteins are chosen from the group consisting of: a.
  • a donor fluorescent protein comprising at least one amino acid substitution at a position corresponding to a position in SEQ ID NO: 1 selected from the group consisting of positions 43, 144, 202, 221 and 227, and an acceptor fluorescent protein corresponding to SEQ ID NO: 4;
  • a donor fluorescent protein corresponding to SEQ ID NO: l and an acceptor fluorescent protein comprising at least one amino acid substitution at a position corresponding to a position in SEQ ID NO: 4 selected from the group consisting of positions 39, 144, 145 and
  • a donor fluorescent protein comprising at least one amino acid substitution at a position corresponding to a position in SEQ ID NO: 1 selected from the group consisting of positions 43, 144, 202, 221 and 227, and an acceptor fluorescent protein comprising at least one amino acid substitution at a position corresponding to a position in SEQ ID NO: 4 selected from the group consisting of positions 39, 144, 145 and 196;
  • the at least one amino acid substitution is selected from the group consisting of T43E, T43D, N144E, N144D, S202E, S202D, L221A, A227E and A227D in SEQ ID NO: l,and from the group consisting of E39R, E144K, E144R, A145K, A145R, N196K and N196R in SEQ ID NO: 4.
  • amino acid mutations described herein can be substituted for other amino acid mutations at the specific residue or residues (or a nearby residue or residues) described herein, that achieves the same effect as the novel fluorescent proteins described herein (e.g., increased FRET). It is well within the level of those of ordinary skill in the art to create a pair of fluorescent proteins based on the mutated amino acid residues of the present invention to create additional pairs of fluorescent proteins aside from the ones described herein. Therefore, additional pairs of fluorescent proteins containing an amino acid other than those described herein are encompassed by the instant invention. Further (non-limiting) candidate amino acids are given in Table 7, which is based on the result of the filtering steps described above.
  • a novel pair of fluorescent proteins wherein one or both of the fluorescent proteins are having an amino acid mutation at a position as described herein wherein the position is the site for mutation but the amino acid may differ, is encompassed by the present invention.
  • the mutation at position N144 in SEQ ID NO: 1 is not required to be N144E or N144D, but can be any other mutation as long as the properties of the engineered fluorescent protein pair are not altered or otherwise are enhanced.
  • homologs of the donor or acceptor fluorescent proteins encompass polypeptides having amino acid substitutions, deletions and/or insertions, preferably by a conservative change, relative to the unmodified protein in question, and having similar biological and functional activity as the unmodified protein from which they are derived; or in other words, without significant loss of function or activity, (for example, homologous fluorescent proteins derived from one species, e.g. Aequorea GFP, either naturally occurring or made by man).
  • homologous fluorescent proteins derived from one species e.g. Aequorea GFP, either naturally occurring or made by man.
  • Percentage similarity and identity can be determined electronically. Examples of useful algorithms are PILEUP (Higgins & Sharp, CABIOS 5:151 (1989), BLAST and BLAST 2.0 (Altschul et al. J. Mol. Biol. 215: 403 (1990). Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (http://www/ncbi.nlm. nih.gov/).
  • a homolog has a sequence identity at protein level of at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, preferably at least 90%, even more preferably at least 95%, at least 98%, at least 99%, as measured in a BLASTp or any other equivalent method known in the art.
  • the donor and the acceptor fluorescent proteins comprised in the pair are each fused to a molecule of interest, preferably a polypeptide of interest or a target polypeptide, optionally through a linker molecule.
  • a "polypeptide of interest” or a “target protein” or grammatically equivalents thereof, as used herein, can be any polypeptide, including, for example, a sensor protein such as calmoduline, or a cellular polypeptide such as an enzyme, a G-protein, a growth factor receptor, or a transcription factor, amongst others, and can be one or two or more proteins that can interact or associate to form a complex.
  • the engineered donor and acceptor fluorescent protein of the present invention may each be linked to a molecule of interest either directly or indirectly, using any linkage that is stable under the conditions to which the protein- molecule complex is to be exposed.
  • the molecule of interest is a polypeptide
  • a convenient means for linking or fusing a fluorescent protein variant and the molecule is by expressing them as a fusion protein from a recombinant nucleic acid molecule, which comprises a polynucleotide encoding, for example, a fluorescent protein operably linked to a polynucleotide encoding the polypeptide molecule.
  • linker molecules are peptides of 1 to 50 amino acids length and are typically chosen or designed to be unstructured and flexible. These include, but are not limited to, synthetic peptides rich in Gly, Ser, Thr, Gin, Glu or further amino acids that are frequently associated with unstructured regions in natural proteins [31]. Examples used herein are (GS)s (SEQ ID NO:31) or (GS)i 0 (SEQ ID NO: 32)or a 50 amino acid randomized sequence of Gly, Serjhr, Gin, Glu (SEQ ID NO: 33).
  • a protease cleavage site such as Factor Xa cleavage site having the sequence IEG (SEQ ID NO:34), the thrombin cleavage site having the sequence LVPR (SEQ ID NO:35), the enterokinase cleaving site having the sequence DDDDK (SEQ ID NO:36), or the PreScission cleavage site LEVLFQGP (SEQ ID NO: 37).
  • the amino acid linker sequence is relatively short, but long enough to allow the contact of enhanced donor and acceptor fluorescent proteins, and does not interfere with the biological activity of the proteins.
  • linker and spacer are used interchangeably herein. Non-limiting examples of suitable linker sequences are also described in the Example section.
  • the invention also provides a fusion protein comprising a polypeptide of interest fused to an engineered donor fluorescent protein or to an engineered acceptor fluorescent protein, as described hereinbefore. More specifically, the present invention also encompasses a "bimolecular construct" of two such fusion proteins, wherein one fusion protein comprises a polypeptide of interest fused to an engineered donor fluorescent protein and one other fusion protein comprises a polypeptide of interest fused to an engineered acceptor fluorescent protein. It should be understood that bimolecular constructs refer to two separate fusion proteins.
  • the bimolecular constructs as described herein may be expressed from a single recombinant nucleic acid molecule or from two separate recombinant nucleic acid molecules, as described further herein.
  • Such a bimolecular construct is particularly useful for detection of protein-protein interactions, which, in turn, can serve as indicator of changes in protein signaling, protein modifications or protein localization.
  • the invention provides a bimolecular construct comprising: a. a donor fluorescent protein fused to a polypeptide of interest, optionally through one or more linker molecules, and
  • an acceptor fluorescent protein fused to a polypeptide of interest, optionally through one or more linker molecules, and wherein said donor and acceptor fluorescent proteins are comprised in the pair of engineered fluorescent proteins as described herein above.
  • a fusion protein comprising one or more polypeptide(s) of interest, an engineered donor and an acceptor fluorescent protein as described hereinbefore, which is then referred to as a "unimolecular construct" or "tandem construct".
  • the different polypeptides can be fused to each other in any order and either directly or indirectly through one or more linker molecules.
  • Such a unimolecular construct can be particularly useful for the detection of conformational changes or intra-molecular binding within a polypeptide of interest which, in turn, can serve as proxy for changes in cellular signaling, ion concentrations or enzymatic activities.
  • Unimolecular constructs are also used for the detection of peptide cleavage events, in particular, due to protease activities.
  • the invention envisages a unimolecular protein construct selected from the group comprising a fusion protein construct as follows: a. a donor fluorescent protein - a polypeptide of interest - an acceptor fluorescent protein, optionally fused through one or more linker molecules, or
  • polypeptide of interest optionally fused through one or more linker molecules, or d.
  • a polypeptide of interest an acceptor fluorescent protein - a donor fluorescent protein
  • the polypeptides comprised in any of the above described fusion proteins can be linked through peptide bonds.
  • the fusion proteins may be expressed from a recombinant nucleic acid molecule containing a polynucleotide encoding an engineered fluorescent protein of the invention operatively linked to one or more polynucleotides encoding one or more polypeptides of interest.
  • the present invention also provides an engineered fluorescent protein comprising at least one amino acid substitution at a position corresponding to a position in SEQ ID NO: 1 selected from the group consisting of positions 43, 144, 202, 221 and 227, such as T43E, T43D, N144E, N144D, S202E, S202D, L221A, A227E and A227D.
  • the present invention also provides an engineered fluorescent protein comprising at least one amino acid substitution at a position corresponding to a position in SEQ ID NO: 4 selected from the group consisting of positions 39, 144, and 145, such as E39 , E144K, E144R, A145K, A145R, N196K and N196R.
  • a fusion protein comprising a polypeptide of interest fused to the engineered fluorescent protein as described hereinbefore, optionally through a linker molecule, is also envisaged here.
  • the invention relates to one, two, or more polynucleotides encoding the engineered donor and/or the acceptor fluorescent proteins, or the engineered fluorescent proteins, or the bimolecular or unimolecular constructs, or the fusion proteins, all as described hereinbefore.
  • Non- limiting examples of polynucleotide sequences encoding engineered fluorescent protein variants and synthetic protein constructs according to the invention, as well as the corresponding amino acid sequences, are listed in Table 8.
  • the invention further concerns expression vectors containing such polynucleotides and host cells containing such polynucleotides or vectors.
  • the vector generally contains elements required for replication in a prokaryotic or eukaryotic host system or both, as desired.
  • Such vectors which include plasmid vectors and viral vectors such as bacteriophage, baculovirus, retrovirus, lentivirus, adenovirus, vaccinia virus, semliki forest virus and adeno-associated virus vectors, are well known and can be purchased from a commercial source (e.g. Promega, Madison Wl; Stratagene, La Jolla CA; GIBCO/B L, Gaithersburg MD) or can be constructed by one skilled in the art.
  • the viral vector can be selected based on its ability to infect one or few specific cell types with relatively high efficiency.
  • the viral vector also can be derived from a virus that infects particular cells of an organism of interest, for example, vertebrate host cells such as mammalian host cells.
  • Viral vectors have been developed for use in particular host systems, particularly mammalian systems and include, for example, retroviral vectors, other lentivirus vectors such as those based on the human immunodeficiency virus (HIV), adenovirus vectors, adeno-associated virus vectors, herpesvirus vectors, vaccinia virus vectors, and the like.
  • HIV human immunodeficiency virus
  • adenovirus vectors adeno-associated virus vectors
  • herpesvirus vectors vaccinia virus vectors, and the like.
  • an antibody that specifically binds to an engineered fluorescent protein according to the invention is also envisaged here.
  • a fourth aspect of the invention is drawn to a kit used for making and using a pair of engineered fluorescent proteins according to the invention in laboratory methods or other applicable uses, including, for example, to construct a fluorescent fusion protein comprising a fluorescent protein of the invention that can be expressed in living cells, tissues, and organisms.
  • the invention provides kits comprising any of the above described polynucleotides or any of the above described expression vectors.
  • the kits can provide any of the above described (pair of) engineered fluorescent proteins or fusion proteins themselves.
  • kits of the present invention can also include, for example but not limited to, apparatus and reagents for sample collection and/or purification, apparatus and reagents for product collection and/or purification, reagents for bacterial cell transformation, reagents for eukaryotic cell transfection, previously transformed or transfected host cells, sample tubes, holders, trays, racks, dishes, plates, instructions to the kit user, solutions, buffers or other chemical reagents, suitable samples to be used for standardization, normalization, and/or control samples. Also present in the kits may be antibodies specific to the provided proteins.
  • kits may be packaged in combination or alone in the same or in separate containers, depending on, for example, cross-reactivity or stability, and can also be supplied in solid, liquid, lyophilized, or other applicable form.
  • a container means of the kits may generally include, for example, at least one vial, test tube, flask, bottle, syringe or other container means, into which a component may be placed, and preferably, suitably aliquoted. Such a kit may be useful for any of the applications of the present invention as described further herein.
  • a fifth aspect of the invention relates to the use of any of the engineered fluorescent proteins (whether as a pair or individual, and whether in a bimolecular or unimolecular construct) or any polynucleotide encoding such protein in any method that employs a fluorescent protein.
  • the engineered fluorescent proteins are useful as a FRET pair for in vitro and/or in vivo FRET-based applications, such as detection of protein-protein interactions, conformational changes of a protein, protease activity, protein modifications, changes in concentrations of metabolites, ions or signaling molecules.
  • the aforementioned FRET-based assay is part of a screening process, for example, in order to discover or characterize compounds, conditions or processes that trigger or disrupt protein-protein interactions or that lead to other changes in the state of cellular or in-vitro systems.
  • fluorescent protein variants having features of the invention are useful as a FRET pair in a method of identifying a specific interaction of a first molecule and a second molecule, for example a specific interaction between proteins, or between a protein and a nucleic acid, or between nucleic acids.
  • the first and second molecules can be cellular proteins that are being investigated to determine whether the proteins specifically interact, or to confirm such an interaction.
  • Such first and second cellular proteins can be the same, where they are being examined, for example, for an ability to oligomerize, or they can be different where the proteins are being examined as specific binding partners involved, for example, in an intracellular pathway.
  • the first and second molecules also can be a polynucleotide and a polypeptide, for example, a polynucleotide known or to be examined for transcription regulatory element activity and a polypeptide known or being tested for transcription factor activity.
  • the first molecule can comprise a plurality of nucleotide sequences, which can be random or can be variants of a known sequence, that are to be tested for transcription regulatory element activity
  • the second molecule can be a transcription factor, such a method being useful for identifying novel transcription regulatory elements having desirable activities.
  • the conditions for such an interaction can be any conditions under which is expected or suspected that the molecules specifically interact.
  • the molecules to be examined are cellular molecules, the conditions generally are physiological conditions.
  • the method can be performed in vitro using conditions of buffer, pH, ionic strength, and the like, that mimic physiological conditions, or the method can be performed in a cell or using a cell extract.
  • the invention envisages a method of identifying a specific interaction of a first polypeptide of interest and a second polypeptide of interest, the method comprising: a. Contacting the first polypeptide of interest, which is fused to a donor fluorescent protein of a pair of engineered fluorescent proteins according to the invention, and the second polypeptide of interest, which is fused to the corresponding acceptor fluorescent protein of the pair of engineered fluorescent proteins according to the invention, under conditions that allow a specific interaction of the first and the second polypeptide of interest,
  • the first polypeptide is a first cellular protein or fragment thereof and the second polypeptide is a second cellular protein or fragment thereof,.
  • the use of the fluorescent protein variants of the invention for such a purpose provides a substantial advantage in that (1) the FRET signal is increased over noise and background, (2) FRET signals can be obtained without optimizing the distance and orientation between the interacting proteins of interest and the donor and acceptor probes which, (3) allows to use standardized long (rather than customized short) molecular linkers between proteins of interest and fluorescent probes, which (4) diminishes the risk of artifacts or modification of the biological activity of interest.
  • the above processes can be miniaturized and automated to enable screening many thousands of molecules in a high throughput format.
  • the engineered fluorescent proteins are useful as a FRET pair to detect cleavage of a substrate having the donor and acceptor coupled to the substrate on opposite sides of the cleavage site.
  • the donor/acceptor pair physically separate, eliminating FRET.
  • Such an assay can be performed, for example, by contacting the substrate with a sample, and determining a qualitative or quantitative change in FRET.
  • a fluorescent protein variant donor/acceptor pair also can be part of a fusion protein coupled by a peptide having a proteolytic cleavage site.
  • useful applications include F ET-based sensors for protein kinase and phosphatase activities or indicators for small ions and molecules such as Ca 2+ , Zn 2+ , cyclic 3', 5'- adenosine monophosphate, and cyclic 3', 5' -guanosine monophosphate.
  • the fluorescent protein variants are useful as fluorescent markers in the many ways fluorescent markers already are used, including, for example, coupling fluorescent protein variants to antibodies, polynucleotides or other receptors for use in detection assays such as immunoassays or hybridization assays, or to track movement of proteins in cells.
  • fluorescent protein variants can be used as labeling substance, include, without the purpose of being limitative, biological and/or medicinal imaging, fluorescent microscopy, FRET-based assays or screening or imaging (in vitro, in cells or in vivo).
  • Fluorescence in a sample generally is measured using a fluorimeter and methods of performing assays on fluorescent materials are well known in the art (see, for example, Lakowicz, “Principles of Fluorescence Spectroscopy” (Plenum Press 1983); Herman, “Resonance energy transfer microscopy” In “Fluorescence Microscopy of Living Cells in Culture” Part B, Meth. Cell Biol. 30:219-243 (ed. Taylor and Wang; Academic Press 1989); Turro, "Modern Molecular Photochemistry” (Benjamin/ Cummings Publ. CoJ, jfric. 1978), pp. 296-361, each of which is incorporated herein by reference).
  • the sample to be examined can be any sample, including a biological sample, an environmental sample, or any other sample for which it is desired to determine whether a particular molecule is present therein.
  • the sample includes a cell or an extract thereof.
  • the cell can be obtained from a vertebrate, including a mammal such as a human, or from an invertebrate, and can be a cell from a plant or an animal.
  • the cell can be obtained from a culture of such cells, for example, a cell line, or can be isolated from an organism.
  • the cell can be contained in a tissue sample, which can be obtained from an organism by any means commonly used to obtain a tissue sample, for example, by biopsy of a human. Where the method is performed using an intact living cell or a freshly isolated tissue or organ sample, the presence of a molecule of interest in living cells can be identified, thus providing a means to determine, for example, the intracellular compartmentalization of the molecule.
  • FP Citrine [10] and mCherry [11] are among the most widely used fluorescent protein (FP) variants.
  • the yellow FP Citrine [10] is derived from Aequorea victoria GFP. It is assumed to retain the weak homodimerization tendency of GFP but can be converted into a strict monomer (mCitrine) by the interface-breaking mutation A206K [5].
  • mCherry [11] is a monomeric and improved variant of Discosoma red fluorescent protein. As the two fluorophores originate from different species (jelly fish and coral), they show no intrinsic interaction.
  • (m)Citrine / mCherry are an excellent long-wavelength FRET pair combining good spectral separation and single exponential donor decay kinetics with among the longest Forster distance reported for any genetically encoded pair [12].
  • FRET donor constructs all shared the same primary structure architecture and were composed (from N- to C-terminal) of: (1) a FKBP12 domain (SEQ ID NO: 11) preceded by a Thr-Gly spacer, (2) a 10 (SEQ ID NO: 31) or 20 amino acid linker (SEQ ID NO: 32) consisting of 5 or 10 repeats of a Gly-Ser dipeptide and preceded and followed by a Thr-Gly spacer (amounting to an overall linker length of 14 or 24 amino acids), (3) the mCitrine reference or modified sequence followed by a Ser-Gly spacer (see Table 8), (4) a recognition and cleavage site for the PreScission protease (SEQ ID NO: 37) followed by a Thr-Gly spacer, (5) a hexa -Histidine tag for purification purposes followed by a Thr-Gly spacer and STOP codon.
  • a FKBP12 domain SEQ ID NO: 11
  • FRET acceptor constructs all shared the same primary structure architecture and were composed (from N- to C-terminal) of: (1) a FRB domain preceded by a Thr-Gly spacer, (2) a 10 or 20 amino acid linker consisting of 5 or 10 repeats of a Gly-Ser dipeptide and preceded and followed by a Thr-Gly spacer (amounting to an overall linker length of 14 or 24 amino acids), (3) the mCherry reference or modified sequence followed by a Ser-Gly or a Gly-Ser spacer, (4) a recognition and cleavage site for the PreScission protease followed by a Thr-Gly spacer, (5) a hexa-Histidine tag for purification purposes followed by a Thr-Gly spacer and STOP codon. Table 3 lists all proteins constructed for this study.
  • Figure 3 compares FRET efficiencies of different conventional and electrostatically enhanced protein pairs.
  • Example fluorescence spectra and lifetime traces are given in Figure 4.
  • Prolongation of the flexible linker between binding and reporter domains leads to the expected drop in FRET efficiency, from 29.9 ⁇ 0.3 % to 23.7 ⁇ 0.6 %.
  • the conventional FRET signal is not influenced by the monomer-enforcing Citrine A206K mutation.
  • the implementation of the top-ranking electrostatic interaction (4-/6+) increases FRET efficiencies to 49.4 ⁇ 0.8 % and 45.6 ⁇ 0.6 % for short and longer linker, respectively.
  • the lower-ranking variant (4-/5+) yields slightly lower signals albeit still much improved over the conventional FRET pair.
  • the monomer-enforcing Citrine mutation A206K situated in the center of the interface, results in similarly small but significant reductions of all enhanced FRET efficiencies.
  • Table 2 compares FRET efficiencies of the most important FRET pairs characterized with various protocols in bulk samples as well as in an in vitro microscopy setup. Absolute FRET efficiencies are consistent across a variety of measurement methods. In particular, efficiencies obtained from the decrease of amplitude-weighted average lifetimes (x amp ) confirm the results of intensity-based experiments ( Figure 4). Donor fluorescence decay remained monoexponential before the induction of protein-protein interactions, testifying to the absence of background FRET. Post induction, the enhancement of FRET was evident from the shortening of average lifetimes. Interestingly, a simple double exponential was only a poor model for the excited state time course of both conventional and enhanced FRET pairs, indicating substantial freedom for the sampling of different relative orientations and distances. A stretched exponential model [18] accounts for the dynamic averaging over disordered ensembles and resulted in quantitative agreement between intensity and life-time-based FRET efficiencies.
  • Electrostatic potentials in Figure 2 were calculated by numeric solution of the Poisson-Boltzmann equation with the DelPhi v program [38].
  • Donor and acceptor structures were refined in complex by FoldX, separated, hydrogens added with Chimera[25], atom names adapted to Amber conventions and histidine residues renamed according to protonation state (Biskit).
  • DelPhi calculations were performed using 12 points per nm grid spacing, 60% maximum filling of linear lattice dimensions, 0.14 nm probe radius, dielectric constants of 80 (solvent) and 4 (protein interior), Amber partial charges and 0.15 M ionic strength with 0.2 nm ionic radius. Potential maps and surfaces were visualized with the UCSF Chimera package[25].
  • Expression plasmids were transformed into f. coli BL21(DE3) (Invitrogen). Starter cultures (LB, 50 ⁇ g/ml kanamycin) were inoculated from single colonies, grown over night at 37°C and then used for 1:100 inoculation of 0.5 I production cultures (2xTY, 50 ⁇ g/ml kanamycin). Production cultures were grown shaking to an O.D. of around 0.6, induced with 0.5 mM IPTG and incubated over night at 20°C. Cells were harvested by centrifugation for 15 min at 6000 g and 4°C , washed once in 15 ml PBS, weighed and stored at -80°C .
  • Pellets were resuspended in 5 ml/(g pellet) BugBuster lysis buffer (Novagen), supplemented with Complete protease inhibitor(Roche) at 1 tablet/50 ml. The lysis mix was incubated for 20 min slowly shaking at room temperature. Cell debris was removed by 5 min centrifugation at 1500 g at 4°C , followed by 30 min centrifugation at 20,000 g and4°C to remove insoluble protein.
  • the supernatant was mixed with 4 ml Ni-NTA Agarose resin (Qiagen, washed twice), diluted to 40 ml with binding buffer (25 mM Tris-Hcl, 20 mM imidazole, 0.5 M NaCI, 10% glycerol, pH7.4) and incubated rotating for 30 min at 4°C .
  • binding buffer 25 mM Tris-Hcl, 20 mM imidazole, 0.5 M NaCI, 10% glycerol, pH7.4
  • the resin was washed (1 min centrifugation at 2000 g) twice with40 ml washing buffer (25 mM Tris-Hcl, 40 mM imidazole, 1 M NaCI, 10% glycerol, 0.1% Tween 20, pH 7.4),transferred to gravity flow columns (BioRad), settled with 30 ml binding buffer and protein was then eluted by gravity flow with 2 x 1 ml elution buffer (25 mM Tris-Hcl, 0.5 M imidazole, 0.5 M NaCI, 10% glycerol, pH 7.4).
  • Measurements were performed in 150 ⁇ volumes in black flat-bottom 96-well plates with 0.3 ⁇ final donor and 0.5 ⁇ final acceptor concentration (for donor FRET efficiency) or, vice versa, 0.5 ⁇ final donor and 0.3 ⁇ acceptor concentration (for sensitized emission) in HBSP+ buffer (see above) at pH 7.4and 25 to 27°C . All proteins were pre-diluted to 15 ⁇ and then diluted to working solutions of 0.45 ⁇ donor9and 1.5 ⁇ acceptor (donor FRET) or 0.75 ⁇ donor and 0.9 ⁇ acceptor (sensitized emission).
  • F D is the fluorescence intensity of the donor-only sample (D)
  • F AD is the intensity of the donor+acceptor sample (AD) corrected for (in practice negligible) acceptor "bleed-through" F 530 A .
  • efficiency of FRET was also calculated from measurements before and after addition of rapamycin:
  • Equation 3 still needs correction for F 610 ⁇ the (non-negligible) donor fluorescence at Cherry emission wavelengths.
  • F 6 io° is determined from the donor-only measurements (D) but is subject to FRET-based quenching in the mixed (AD) sample:
  • FLIM (fluorescence lifetime imaging)-F ET was measured by time-correlated single-photon counting (TCSPC) with an inverted multiphoton laser scanning microscope (Leica TCS SP5) using a 63x water immersion N.A 1.2 Plan-Apochromat objective, and equipped with a single molecule detection platform and single-photon counting electronics (PicoHarp 300) from PicoQuant GmbH (Berlin).
  • Donor (mCitrine) two-photon excitation was performed at 950nm from a Mai Tai Ti:Sapphire laser (Spectra Physics) with a repetition rate of 80 MHz. Photons were detected by a SPAD set up (PicoQuant).
  • a fluorescence bandpass filter 500-550 nm) limited the detection to the donor fluorescence only.
  • vjherer DA is the amplitude-weighted mean fluorescence lifetime of the donor (mCitrine) in the presence of both acceptor (mCherry) and rapamycin.
  • T D is the mean fluorescence lifetime of the donor (mCitrine) in the presence of acceptor (mCherry) without rapamycin.
  • T D of the donor in the presence of the acceptor but without rapamycin was calculated from a mono-exponential fit to the fluorescence lifetime decays.
  • experimental decay curves were fit to a stretched bi-exponential model [18].
  • the noninteracting protein's lifetime was fixed to r D and the value of r DA and stretching factor ⁇ were estimated.
  • Table 1 ln-silico interface designs
  • V V inserted after Metl so that the mRNA should contain an ideal translational start sequence. We number such a V as la to preserve wild-type numbering for the rest of the sequence.
  • Table 8 Overview amino acid and nucleotide sequences used in this study.
  • Non-limiting examples of reference fluorescent proteins engineered fluorescent protein variants, test protein-protein interaction and synthetic protein constructs
  • X can be amino acid A or K
  • Nucleotide sequences of synthetic protein constructs can be easily derived by combining the nucleotide sequences as provided in SEQ ID NOs: 20-30.

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Abstract

La présente invention se rapporte à des paires de protéines fluorescentes génétiquement modifiées et à leurs utilisations dans des applications à base de FRET, ainsi qu'à des procédés de production de protéines fluorescentes génétiquement modifiées pour FRET hautement efficace (Transfert d'Énergie par Résonance de Type Förster) basés sur l'introduction d'interactions entre auxiliaires électrostatique.
PCT/EP2012/075737 2011-12-15 2012-12-17 Protéines fluorescentes génétiquement modifiées à des fins d'amélioration du fret et leurs utilisations WO2013087921A1 (fr)

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JPWO2016204296A1 (ja) * 2015-06-19 2018-04-05 国立研究開発法人理化学研究所 ｐＨ応答性のタンパク質分解プローブ
US10591486B2 (en) 2014-07-23 2020-03-17 Institut National De La Sante Et De La Recherche Medicale (Inserm) Methods and tools for detecting interactions in eukaryotic cells using microtubule structures and dynamics

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US10591486B2 (en) 2014-07-23 2020-03-17 Institut National De La Sante Et De La Recherche Medicale (Inserm) Methods and tools for detecting interactions in eukaryotic cells using microtubule structures and dynamics
JPWO2016204296A1 (ja) * 2015-06-19 2018-04-05 国立研究開発法人理化学研究所 ｐＨ応答性のタンパク質分解プローブ
EP3312279A4 (fr) * 2015-06-19 2018-11-21 Riken SONDE DE DÉCOMPOSITION DES PROTÉINES SENSIBLE AU pH
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