WO2013087922A1 - Procédés de production de protéines fluorescentes génétiquement modifiées à des fins d'amélioration du fret, produits et leurs utilisations - Google Patents

Procédés de production de protéines fluorescentes génétiquement modifiées à des fins d'amélioration du fret, produits et leurs utilisations Download PDF

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WO2013087922A1
WO2013087922A1 PCT/EP2012/075743 EP2012075743W WO2013087922A1 WO 2013087922 A1 WO2013087922 A1 WO 2013087922A1 EP 2012075743 W EP2012075743 W EP 2012075743W WO 2013087922 A1 WO2013087922 A1 WO 2013087922A1
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donor
pair
protein
acceptor
fret
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Raik GRÜNBERG
Luis Serrano Pubul
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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 WO2013087922A1 publication Critical patent/WO2013087922A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/43504—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates
    • 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
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/60—Fusion polypeptide containing spectroscopic/fluorescent detection, e.g. green fluorescent protein [GFP]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/70—Fusion polypeptide containing domain for protein-protein interaction

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 methods to produce genetically engineered fluorescent proteins for high efficiency FRET (Forster Resonance Energy Transfer). In particular, such methods comprise fusing a donor and acceptor fluorescent protein to a low-affinity helper interaction module. Moreover, pairs of such genetically engineered fluorescent proteins are provided as well as uses thereof in FRET-based applications.
  • 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 may be 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. They are, however, the best way to multiply FRET responses, in particular, if sensors are based on differences between high (bound, connected) and low (unbound, cleaved) local concentrations of donor and acceptor [8].
  • unimolecular FRET constructs can benefit from the weak dimerization of GFP-derived donor and acceptor domains [7, 9].
  • 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 [48].
  • 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.
  • FRET pairs with increased FRET efficiency and less dependency on fluorophore distances and orientations.
  • such pairs (1) would be excited at longer wavelengths in order to reduce phototoxicity and autofluorescence and increase the penetrance of emission light; (2) would be composed of two un-related fluorescent proteins so that heterodimerization would not translate to homodimerization of acceptor or donor; (3) would work by creating a weakly aligned donor/acceptor encounter complex without actual binding, that means, they should show high on rates but very low half live of the donor/acceptor arrangement.
  • helper interactions weak but strictly heterodimeric interactions
  • helper interaction FRET hiFRET
  • One of these methods is based on a modular approach for the rational design of helper interactions between, potentially, any pair of fluorescent proteins.
  • Unmodified FRET probes were aligned through weak domain/peptide interaction modules.
  • the enhanced FRET pairs will be of immediate use both for in vitro or in vivo FRET based applications.
  • signal enhancements through helper interactions should be of particular interest for upcoming large-scale FLIM experiments [22] or applications in thick samples.
  • the modular helper interactions are re-usable for other fluorescent proteins and thus uncouple issues of donor / acceptor orientation and distance from the choice of fluorophores.
  • FRET probe developers can therefore break free from a de-facto limitation to CFP / YFP-derived variants and benefit from the full range of modern fluorescent proteins.
  • FRET probe developers can therefore break free from a de-facto limitation to CFP / YFP-derived variants and benefit from the full range of modern fluorescent proteins.
  • Our helper interactions led to a large increase of FRET efficiency.
  • FRET signals between all synthetic protein constructs were characterized in vitro by donor- and acceptor-based fluorescence intensity.
  • the most promising FRET pairs were validated by lifetime (FLIM) measurements in vitro and in live cells.
  • FLIM lifetime
  • 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 fluorescent protein in FRET measurements,
  • the step of fusing said pair of fluorescent proteins in the above described method is further characterized by a. Fusing the donor fluorescent protein of said pair of fluorescent proteins to one of the constituting polypeptides of said pair of interacting polypeptides, and
  • said fusion may also be an internal fusion, which means an insertion at permissive sites within the donor/acceptor fluorescent protein.
  • said pair of interacting polypeptides can be further characterized by a dissociation constant (K D ) of at least 50 ⁇ .
  • said pair of interacting polypeptides comprises at least one protein domain and at least one peptide specifically interacting with said protein domain, such as for example a. a WW domain or homolog thereof, and a peptide specifically interacting with said WW domain or homolog thereof, or
  • Another aspect of the invention relates to a pair of engineered fluorescent proteins comprising a donor fusion protein and an acceptor fusion protein wherein a.
  • the donor fusion protein comprises a donor fluorescent protein, a first polypeptide, and optionally one or more linkers, and
  • the acceptor fusion protein comprises an acceptor fluorescent protein, a second polypeptide, and optionally one or more linkers, and
  • first and second polypeptide are characterized as a pair of low-affinity interacting polypeptides
  • said pair of engineered fluorescent proteins has increased FRET efficiency relative to the non-engineered pair comprising a donor and acceptor fluorescent protein each of which is not fused to one of the interacting polypeptides.
  • said donor or acceptor fluorescent protein of the above described pair of engineered fluorescent proteins are each fused to said first or second polypeptide at the C- terminal or N-terminal end of said fluorescent protein, optionally through a linker molecule.
  • said donor or acceptor fluorescent protein of the above described pair of engineered fluorescent proteins are each internally fused to said first or second polypeptide, which means an insertion at permissive sites within the donor/acceptor fluorescent protein.
  • said pair of interacting polypeptides are characterized by a dissociation constant (K D ) of at least 50 ⁇ .
  • said pair of interacting polypeptides comprises at least one protein domain and at least one peptide specifically interacting with said protein domain, such as for example a. a WW domain or homolog thereof, and a peptide specifically interacting with said WW domain or homolog thereof, or
  • the above described pair of engineered fluorescent proteins wherein the donor fusion protein and the acceptor fusion protein are each fused to a polypeptide of interest, optionally through one or more linker molecules.
  • the invention provides a bimolecular construct comprising: a. a donor fusion protein of the invention fused to a polypeptide of interest, optionally through one or more linker molecules, and
  • an acceptor fusion protein of the invention fused to a polypeptide of interest, optionally through one or more linker molecules.
  • the invention provides a unimolecular construct selected from the group comprising a fusion protein construct as follows:
  • polynucleotides encoding any of the donor and/or acceptor fusion proteins, or any of the bimolecular/unimolecular constructs, 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.
  • 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 bimolecular/unimolecular constructs, or any of the polynucleotides or any of the expression vectors, all as described hereinbefore, for in vitro and/or in vivo FRET-based applications.
  • the present invention relates to 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 fusion 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 fusion 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,
  • FIG. 1 Design of FRET helper interactions via a modular approach. Fluorophores were aligned by a weak interaction between (a) a WW domain and cognate peptides or (b) a SH3 domain and cognate peptides. Unstructured residues at the mCitrine and mCherry C-terminus were either retained ( ⁇ ) or trimmed ( ⁇ ). Flexible linkers are indicated by a dotted line. Average end-to-end distances ( ⁇ link>) were calculated from the worm-like chain (WLC) polymer model [40] based on the number of unstructured amino acids (aa) and are drawn to scale.
  • WLC worm-like chain
  • FIG. 3 Enhanced FRET, (a) WW - peptide helper interactions with moderate or weak affinity (shades of blue) enhance FRET over conventional probes (gray). The unstructured spacer peptides between FRET and helper modules (-...longer or «... shorter) have only minor effects. The FRB/FKBP12 reference interaction was connected N-terminally via a flexible 24-amino acid linker. Background signal in the unbound state (without rapamycin) remains negligible (at 0.5 ⁇ concentration), (b) Helper interactions based on unrelated SH3 domain - peptide pairs have a similar effect.
  • the -WW / Wp2 helper interaction module leads to substantially increased FRET signals
  • the WW - peptide helper module doubles FRET between mTeal (mTFPl) and mCherry. Constructs were based on the best-performing proteins described in (a) with mCitrine replaced by mTFPl. mCherry bearing a non-cognate SH3 peptide (see b) served as negative control.
  • FIG. 4 Emission spectra (top) and fluorescence lifetime (bottom) of conventional and enhanced FRET pairs.
  • FIG. 9 Comparison of the original (“wt”) and enhanced mCitrine sequences. Domains added to the C-terminal of unmodified mCitrine are shown in green (WW) and blue (SH3). Unstructured peptide sequences introduced during the engineering of the original Citrine are labeled as 'spacer'.
  • FIG. 10 Comparison of the original mCherry sequence with the four variants engineered for domain-peptide recruitment. Only the C-terminal of (the otherwise unmodified) mCherry is shown. Wpl and Wp2 denote strong and weak WW-binding peptides, respectively. Spl and Sp2 denote strong and weak SH3-binding peptides, respectively.
  • EGF epidermal growth factor
  • Donor-intensity images are color-coded with intensity-weighted lifetimes (middle) or intensity-weighted FRET efficiencies (bottom),
  • RBD Ras Binding Domain
  • FIG. 13 Application of conventional and enhanced FRET probes to the detection of the Rafl : BRaf interaction in live HeLa cells, (a) HeLa cells expressing conventional mCitrine fused to the C-terminal of full length Rafl and conventional mCherry coupled to the C-terminal of full-length BRaf are shown untreated (control) or 2 h after treatment with Raf kinase inhibitor GDC-0879. Donor-intensity images (top) are color-coded with non-weighted FRET efficiencies (middle). The distribution of FRET efficiencies over all cells in the field of view is shown in the bottom panel.
  • 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 RNA 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.
  • regulatory sequence 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 NA 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 to the progeny of such a cell.
  • 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 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” is defined here 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” is that change in an amino acid or nucleotide sequences which results from the insertion of 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” generally refers to the insertion or 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.
  • polypeptides that may 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).
  • the terms “specifically interact” or “specifically bind” and grammatical equivalents thereof are used interchangeably herein.
  • 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 high affinity will bind to the available protein domain so as to shift the equilibrium toward high concentration of the resulting complex.
  • 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 (low affinity) 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 strategies 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 preferred strategy is based on the use of weak domain/peptide interaction modules and the implementation for the optimization of fluorescent protein pairs for in vitro/in vivo FRET applications. It is a particular advantageous strategy since (1) the same helper interaction module can be applied to any suitable pair of fluorescent proteins for FRET applications without further engineering, (2) the helper interaction can be easily fine-tuned to the experimental conditions like for example protein concentrations, (3) the helper interaction module can be replaced by another helper interaction module if needed for reasons of assay or host cell compatibility, (4) FRET pairs with different helper interaction modules can be employed in parallel (e.g. in multiplexed assays) without interfering with each other.
  • 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 fluorescent protein 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 [41].
  • fluorescent proteins are often classified according to their spectral class.
  • 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, Renilla reniformis, and Phialidium gregarium [19, 20].
  • 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 [21]. 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 [24, 25] and U.S. Patent No. 5,625,048 and 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 [24], allelic variants thereof, for example, a variant having a Q80R substitution [26]; 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.
  • these fluorescent proteins contain different aromatic amino acids within the central chromophore and fluoresce at a distinctly shorter wavelength than the wild type GFP species.
  • the engineered GFP proteins designated P4 and P4-3 contain, in addition to other mutations, the substitution Y66H; and the engineered GFP proteins designated W2 and W7 contain, in addition to other mutations, Y66W.
  • 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 [11].
  • wild type fluorescent proteins and variants derived thereof are listed in Tables 5 and 6, wherein further details on the properties of each listed protein are provided.
  • 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 [29, 30].
  • 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 non-radioactive 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, and 31] as well as in the Detailed Description further herein.
  • 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 refers to a pair of fluorescent proteins that has been genetically engineered or modified, according to any of the above described engineering methods of the present invention; this is in contrast to a non-engineered fluorescent protein pair that has not been genetically engineered according to the invention, and that is known in the art.
  • a non-engineered pair of fluorescent proteins 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.
  • said 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 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 fluorescent protein in FRET measurements,
  • the fusion of the donor/acceptor fluorescent protein to one of the constituting polypeptides of a pair of low-affinity interacting polypeptides may be either at the C-terminal or at the N-terminal end of said donor/acceptor fluorescent protein, optionally through one or more linker molecules.
  • the fusion of the donor/acceptor fluorescent protein to one of the constituting polypeptides of a pair of low-affinity interacting polypeptides may also be an internal fusion, which means an insertion at permissive sites within the donor/acceptor fluorescent protein.
  • Low-affinity interacting polypeptides may thus also be fused such that they are inserted into permissive sites within the globular fluorescent protein domain.
  • Permissive sites for the insertion of low-affinity interacting peptides are positions within the fluorescent protein where the polypeptide chain can be interrupted without severely affecting the folding or structure of the protein or the maturation of its chromophore. Permissive sites are typically surface-exposed. Circular permutation experiments have revealed many permissive sites within fluorescent proteins where the peptide chain can be opened up [49, and incorporated herein by reference]. Sequence and structure alignments can be used to infer potential permissive sites in one fluorescent protein from experimentally characterized positions in another fluorescent protein variant.
  • low affinity polypeptides should, preferably, be inserted at the other end of the barrel.
  • the low affinity interaction and the (C or N terminally attached) protein interaction of interest can thus pull together donor and acceptor proteins from two opposing ends.
  • the pair of interacting polypeptides can be freely chosen and can be any pair of polypeptides specifically interacting with each other as long as they interact with a low affinity.
  • a pair of low-affinity interacting polypeptides refers to a pair of polypeptides that specifically interact (as defined herein) with each other with a low affinity (as defined herein), and is also referred to as "helper interaction module".
  • helper interaction module refers to a pair of polypeptides that specifically interact (as defined herein) with each other with a low affinity (as defined herein), and is also referred to as "helper interaction module".
  • “low affinity” implies that the dissociation constant for the pair of interacting polypeptides is significantly lower than the protein concentrations used in F ET-based in vitro and/or in vivo applications, as elaborated further herein.
  • the dissociation constant should be lower than the target protein concentration by a factor of 19, preferably a factor of 33, most preferably a factor of 99, respectively. Even more preferably, the dissociation constant is lower than the target protein concentration by a factor higher than 100. It should be clear that intracellular protein concentrations are usually below 0.001 mM and very rarely above 0.01 mM, even for the most strongly expressed proteins [37].
  • said pair of interacting polypeptides is characterized by a dissociation constant (K D ) in the range of 10 s M, preferably at least 0.05 mM, more preferably at least 0.1 mM, at least 0.2 mM, at least 0.3 mM, at least 0.5 mM, but not higher than 5 mM, preferably at most 2 mM, or more preferably at most 1 mM.
  • K D dissociation constant
  • said pair of low-affinity interacting polypeptides comprises at least one protein domain and at least one peptide specifically interacting with said protein domain.
  • Domain/peptide interacting polypeptides are well-known in the art, and without the purpose of being limitative, are as described in [36]. Particular examples include protein domains such as WW, SH3, PDZ, PTB, EVH1, GYF, VHF, amongst many others, and peptides specifically binding to said domains, amongst many others.
  • said pair of interacting polypeptides in any of the above described methods is chosen from the group comprising: a. a WW domain or homolog thereof, and a peptide specifically interacting with said WW domain or homolog thereof, or b. an SH3 domain or homolog thereof, and a peptide specifically interacting with said SH3 domain or homolog thereof.
  • homolog of a protein domain/peptide encompasses polypeptides having amino acid substitutions, deletions and/or insertions, preferably by a conservative change, relative to the unmodified polypeptide in question. Homologs of domains/peptides are capable of specifically interacting with each other with different degrees of low affinity (as defined hereinbefore). Homologs may be either naturally occurring or made by man. Several different methods are known by those of skill in the art for identifying and defining these functionally homologous sequences, including phylogenetic methods, sequence similarity and hybridization methods. Percentage similarity and identity can be determined electronically.
  • a homolog of a protein domain/peptide has a sequence identity at amino acid 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.
  • a homolog of a protein domain/peptide can also be identified by sharing the same fold; that means, by having a three-dimensional structure that is significantly similar.
  • Structural homologs can be identified and defined by using structure alignment programs, which are known to those skilled in the art. Examples of structure alignment algorithms are Dali and Dali server [42] as well as Tm-align [43].
  • a structure homolog of a protein domain is identified by a TM-align score of at least 0.5, at least 0.6, at least 0.7, preferably higher than 0.7.
  • any naturally or engineered fluorescent protein including a variant of a fluorescent protein, can be chosen as a donor or acceptor fluorescent protein, as long as together they are suitable for FRET measurements, as was described hereinbefore.
  • Other examples are provided in Table 5 and 6.
  • the term "variant" as defined hereinbefore encompasses homologs of the donor or acceptor fluorescent proteins.
  • Homologs of a protein 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.
  • a homolog of a donor or acceptor fluorescent protein 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 acceptor fluorescent protein of the pair of fluorescent proteins do not show initial intrinsic interaction, such as by choosing fluorescent protein variants that have been made monomeric, such as for example, without the purpose of being limitative, mCitrine, mCherry, mTFPl.
  • a second aspect of the present invention relates to a pair of engineered fluorescent proteins comprising a donor fusion protein and an acceptor fusion protein wherein a.
  • the donor fusion protein comprises a donor fluorescent protein, a first polypeptide, and optionally one or more linkers, and
  • the acceptor fusion protein comprises an acceptor fluorescent protein, a second polypeptide, and optionally one or more linkers, and
  • first and second polypeptide are characterized as a pair of low-affinity interacting polypeptides
  • said pair of engineered fluorescent proteins has increased FRET efficiency relative to the non-engineered pair comprising a donor and acceptor fluorescent protein each of which is not fused to one of the interacting polypeptides.
  • the donor or acceptor fluorescent protein may be fused to said first or second polypeptide at the C-terminal or N-terminal end of said fluorescent protein, optionally through a linker molecule.
  • the fusion of the donor or acceptor fluorescent protein to said first or second polypeptide may also be an internal fusion, which means an insertion at permissive sites within the donor or acceptor fluorescent protein.
  • the donor fusion protein and the acceptor fusion comprised in the pair of engineered fluorescent proteins are each fused to a molecule of interest, preferably a polypeptide of interest or a target polypeptide, optionally through one or more 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 fusion proteins 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 an engineered fluorescent protein 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 [33]. Examples used herein are (GS)s (SEQ ID NO: 69) or (GS)i 0 (SEQ ID NO: 70) or a 50 amino acid randomized sequence of Gly, Serjhr, Gin, Glu (SEQ ID NO: 71, SEQ ID NO: 98).
  • a protease cleavage site such as Factor Xa cleavage site having the sequence IEG (SEQ ID NO: 72), the thrombin cleavage site having the sequence LVPR (SEQ ID NO: 73), the enterokinase cleaving site having the sequence DDDDK (SEQ ID NO: 74), or the PreScission cleavage site LEVLFQGP (SEQ ID NO: 75).
  • a polypeptide of interest may be fused to an engineered donor or acceptor fusion protein, either at the free N- or C-terminal end of the donor or acceptor fluorescent protein or otherwise at the free N- or C-terminal end of the low-affinity interacting polypeptides.
  • donor and acceptor fusion proteins are constructed themselves; more specifically, whether the donor/acceptor fluorescent protein are each fused to one of the constituting interacting polypeptides either at the C-terminal or at the N-terminal end of said donor/acceptor fluorescent protein.
  • 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.
  • 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 fusion protein or to an engineered acceptor fusion protein, as described hereinbefore.
  • 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 fusion protein and one other fusion protein comprises a polypeptide of interest fused to an engineered acceptor fusion protein.
  • 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 envisages a bimolecular construct comprising: a. a donor fusion protein fused to a polypeptide of interest, optionally through one or more linker molecules, and
  • acceptor fusion protein fused to a polypeptide of interest, optionally through one or more linker molecules, and
  • donor and acceptor fusion proteins are comprised in the pair of engineered fluorescent proteins as described hereinbefore.
  • a fusion protein comprising one or more polypeptide(s) of interest, an engineered donor and an acceptor fusion protein as described hereinbefore, which is then refered 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 construct selected from the group comprising a fusion protein construct as follows:
  • donor and acceptor fusion proteins are comprised in the pair of engineered fluorescent proteins as described hereinbefore.
  • the polypeptides comprised in any of the above described fusion protein constructs can be linked through peptide bonds, as described hereinbefore.
  • 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 invention relates to one, two, or more polynucleotides encoding the engineered donor and/or acceptor fusion proteins of the pair of 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 proteins and synthetic protein constructs according to the invention, as well as the corresponding amino acid sequences, are listed in Table 7.
  • 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.
  • a further 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 an engineered 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.
  • Still another 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.
  • engineered fluorescent proteins 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.
  • 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, and 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 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 fusion protein of the pair of engineered fluorescent proteins of the invention, and the second polypeptide of interest, which is fused to the corresponding acceptor fusion protein of the pair of engineered fluorescent proteins of 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 engineered fluorescent proteins of the present 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 FRET-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.
  • mCitrine [10] and mCherry [11] were used as our reference donor and acceptor fluorophores for the comparison of conventional and enhanced FRET.
  • 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].
  • addition of rapamycin triggers a high-affinity binding between the two domains yet there is no interaction in absence of the drug [14]. This allowed us to measure FRET signals both in bound (active, induced) and unbound state.
  • FRET donor constructs all shared the same primary structure architecture and were composed (from N- to C-terminal) of: (1) a FKBP12 domain preceded by a Thr-Gly spacer, (2) a 20 amino acid linker consisting of 10 repeats of a Gly-Ser dipeptide and preceded and followed by a Thr-Gly spacer (amounting to an overall linker length of 24 amino acids), (3) the mCitrine reference or modified sequence followed by a Ser-Gly 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.
  • 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 20 amino acid linker consisting of 10 repeats of a Gly-Ser dipeptide and preceded and followed by a Thr-Gly spacer (amounting to an overall linker length of 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.
  • the mCitrine sequence (3 above) was replaced by the mCitrine sequence (full length or C- terminally trimmed) followed by a Ser-Gly spacer, followed by the enhancement domain (WW or SH3) followed by a Thr-Gly spacer.
  • the mCherry sequence (3 above) was replaced by the mCherry sequence (full length or C-terminally trimmed) followed by a Ser-Gly or a Gly-Ser spacer, followed by the enhancement peptide of 6 to 11 amino acids length with varying affinities for either WW or SH2 domain followed by a Thr-Gly spacer.
  • FIG. 2a we fused a stabilized (L30K) [15] variant of the WW domain of human YAP65 to the C terminal of mCitrine. Affinities of cognate peptides reported for this domain [15] range from (K D ) 40 to 700 ⁇ . We fused either the strongest or a weakly binding peptide to the C terminal of mCherry. Peptide sequences and affinities are listed in Table 1. The N- and C terminal of all peptide sequences were flanked by Gly-Ser (GS) and Thr-Gly (TG), respectively, in order to prevent spurious interactions with neighboring residues.
  • GS Gly-Ser
  • TG Thr-Gly
  • the conventional mCitrine as well as mCherry contain an additional short unstructured C terminal spacer sequence (GMDELYK) originally introduced for better compatibility with protein fusions. Judging from structural models, the WW / peptide interaction appeared compatible with a tight coupling and the deletion of this spacer sequence should bring donor and acceptor into even closer proximity (see Figure 2a for distances).
  • GMDELYK additional short unstructured C terminal spacer sequence
  • SH3 domain / peptide module The enhancement of FRET probes through domain-based helper interactions is a general design concept. To prove this point, we replicated the same design using an unrelated SH3 domain / peptide interaction module (Figure 2b).
  • Figure 2b We paired the SH3 domain from Saccharomyces cerevisiae protein Shol [16, 17] with two different peptides, chosen to have a stronger or weak affinity similar to the two WW / peptide pairs (Table 1).
  • the SH3 domain was fused to mCitrine and peptides were fused to mCherry. As before, each construct was prepared with and without the MDELYK spacer sequence. Results from in vitro measurements are given in Figure 3b.
  • Table 2 compares FRET efficiencies of the most important FRET pairs characterized with various protocols in bulk samples as well as in a 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. 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.
  • Figure 3c demonstrates the application of the domain / peptide enhanced FRET probes in live cells.
  • WW - peptide co-recruitment doubled the FLIM-FRET signal.
  • no background signal was apparent before the addition of rapamycin.
  • EXAMPLE 6 Direct detection of H-Ras / Rafl interactions
  • Activation of the small GTPase HRas leads to the binding of Rafl (mediated by the RasGTP binding domain, RBD, contained within Rafl) and the recruitment of the complex to the membrane where further signalling events are triggered [38].
  • RBD is located near the N- terminal of Rafl which rules out C-terminal attachment of FRET probes.
  • An unstructured region of 50 amino acids length separates the RasGTP binding domain also from the N-terminal of the protein.
  • N-terminal of the binding domain itself is pointing away from the interacting HRas. Any protein fusion will thus place the fluorescent probe remote from the interacting HRas. It is thus not surprising that no high performance bi-molecular FLIM/FRET probe has so far been described for the physiologically relevant interaction between HRas and full-length Rafl.
  • the enhancement with helper interactions improves the HRas/HRafl FRET sensor to approximately the same level of performance as observed for the classic truncated HRas/RaflRBD construct.
  • the enhanced HRas/HRafl FRET sensor is a biologically more relevant model for the interaction, as the lack of regulatory sequences and the effective de-localization of the RaflRBD protein fragment may lead to non-physiological artifacts in more detailed aspects of signaling.
  • the approximate time course of FRET signals suggests a delayed activation of HRas/RaflRBD sensors which could be tentatively explained by a slow exchange of RaflRBD fragments between cytosolic and non-physiological nuclear fractions.
  • this example demonstrates the straightforward recovery of intracellular high-performance FRET signals from a transient and dynamically regulated protein-protein interaction with a molecular geometry that would be unfavorable for conventional FRET probe design.
  • Rafl also described as CRaf
  • BRaf are part of the Ras-ERK MAPK signaling pathway (see Example 6). This pathway is often involved in the malignant proliferation of human cancer cells, for example, 43% of melanomas are characterized by oncogenic mutations activating BRaf [46].
  • BRaf is a downstream effector of activated H-Ras. Inhibitors of Raf kinase activity were therefore considered promising antitumor agents as they should block proliferation signals both from oncogenic BRaf as well as from mutated H-Ras.
  • Raf kinase inhibitors failed to act against tumors driven by oncogenic Ras. Furthermore, therapeutic success against oncogenic BRaf is often limited by the rapid development of drug resistance. Both failures have recently been traced back to activating interactions between Rafl and BRaf [45-47]. In the presence of activated Ras, Raf kinase inhibitors turned out to promote the heterodimerization of BRaf with Rafl leading to the activation of downstream proliferation signals by Rafl kinase thus circumventing the block of BRaf kinase. Depending on cellular context (oncogenic Ras instead of oncogenic BRaf), these drugs can thus promote rather than inhibit cancer growth.
  • Rafl / BRaf interaction has until now not been visualized in live cells.
  • Existing assays are biochemical (immunoblots of phosphorylated substrate proteins or co- immunoprecipitation of Rafl and BRaf), and are performed in cell lysates.
  • Caspase 3 Activation of executioner Caspase 3 is a crucial event in programmed cell death (apoptosis).
  • Caspase 3 is a tightly regulated protease that recognizes a specific cleavage sequence (DEVD).
  • DEVD specific cleavage sequence
  • FRET sensors allow the detection of Caspase 3 activity, and thus initiation of apoptosis in life cells [50].
  • Caspase sensors are simple unimolecular construct where a donor fluorescent protein is fused via a short linker to an acceptor fluorescent protein resulting in a strong FRET signal between the two. The short linker contains the specific Caspase cleavage sequence. Upon activation of Caspase 3, this linker is cleaved and the FRET signal is rapidly lost.
  • the cleavage can be followed by simply measuring the ratio of acceptor (sensitized emission) versus donor fluorescence since the stochiometry of both is fixed.
  • Known Caspase sensors perform well and are difficult to improve on.
  • simple proteolytic sensors such as this have often been used to evaluate or optimize new donor / acceptor FRET pairs [4].
  • helper interactions were fused to the mTurquoise2 N-terminal (WW domain) and Citrine C-terminal (Wp2 peptide) (SEQ ID NO: 89) and measured an improved emission ratio of 1.99 ⁇ 0.28 switching to 0.79 ⁇ 0.07 after apoptosis.
  • WW domain WW domain
  • Wp2 peptide Citrine C-terminal
  • helper interaction modules can also be inserted within the fluorescent protein structure.
  • Preferred positions of insertion are sites such as Gly 174 which had previously been shown to tolerate circular permutation [49]. Further optimization of insertion site and surrounding sequences may help to mitigate effects on fluorophore maturation and folding.
  • helper interaction modules can be robustly attached at either N- or C-terminal or even inside of different fluorescent proteins and can lead to significant improvements of ratiometric FRET measurements.
  • Protein-coding constructs were obtained by gene synthesis from DNA 2.0 (CA, USA) and delivered in expression plasmid pJExpress411, codon-optimized for E.coli.
  • pJExpress411 codon-optimized for E.coli.
  • selected constructs were transferred from the pJExpress411 into a pcDNA3.1 vector backbone using In-Fusion (Clonetech) or isothermal assembly [39] recombining.
  • the appropriate PC reactions were performed with Phusion Hotstart polymerase (NEB). All constructs were verified by DNA sequencing.
  • H-Ras, Rafl, Rafl RBD, BRaf or Caspase3 sensors were again obtained by gene synthesis from DNA 2.0 and delivered in pJ603-neo (mCitrine and Caspase constructs) or pJ609-puro (mCherry constructs) mammalian expression vectors and codon-optimized for Homo sapiens.
  • sequences encoding Caspase sensors were transferred from pJ603-neo into the pJExpress411 vector backbone, including C terminal fusion to PreScission recognition site and hexa-Histidine tag, using PCR and isothermal assembly [39]. Protein expression and purification
  • Expression plasmids were transformed into E. 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 and 4°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, pH 7.4) and incubated rotating for 30 min at 4°C .
  • binding buffer 25 mM Tris-Hcl, 20 mM imidazole, 0.5 M NaCI, 10% glycerol, pH 7.4
  • the resin was washed (1 min centrifugation at 2000 g) twice with 40 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).
  • 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 61 ⁇ , the (non-negligible) donor fluorescence at Cherry emission wavelengths.
  • F 61 o° is determined from the donor-only measurements (D) but is subject to FRET-based quenching in the mixed (AD) sample:
  • Overall standard deviations for E were determined by error propagation. Standard deviations reported for E A do not include the error arising from the determination of extinction coefficients ⁇ 516 ⁇ and s 516 D (Table 4). Owing to these additional uncertainties, E A is unlikely to perfectly agree with E D but serves as a consistency check. Large deviations of E A would indicate errors in protein concentrations or systematic defects in binding or fluorescent domains.
  • Recombinant human Caspase 3 was purchased from R&D Systems, MN. Caspase reactions were performed in 150 ⁇ volumes in black flat-bottom 96-well plates with 0.5 ⁇ FRET sensor concentration in the buffer recommended by the manufacturer manufacturer (25 mM HEPES pH 7.5, 10 mM DTT, 0.1 % CHAPS) at 30 °C . Donor fluorescence (excitation 434 nm, emission 474 nm) and sensitized emission (excitation 434, emission 529 nm) were measured on a Tecan M1000 plate reader with 5 nm bandwidth for excitation and emission. 5 ⁇ Caspase 3 containing a total of 50 ng enzyme (or buffer as control) were then added to each well simultaneously. After short initial shaking, donor and sensitized emission signals were recorded every 30 s for 40 min. ln-vitro FLIM
  • FLIM (fluorescence lifetime imaging)-FRET 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 ThSapphire 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.
  • Mean FRET efficiency values, E were calculated from: where r DA is the amplitude-weighted mean fluorescence lifetime of the donor (mCitrine) in the presence of both acceptor (mCherry) and rapamycin. r 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. Under FRET conditions, experimental decay curves were fit to a stretched bi-exponential model [18]. The non-interacting protein's lifetime was fixed to T D and the value of T DA and stretching factor ⁇ were estimated.
  • HeLa cells were cultured at 37°C and 5% C02 in DMEM(Gibco), supplemented with 10% FBS, 1% glutamate, and 1% pen/strep. After 48 h, 1x10 s cells were seeded on glass-bottomed 6-cm cell culture plates (MatTek Corp.) and grown overnight. Cells were transfected with 4 ⁇ g of donor only (control) or double transfected with 4 ⁇ g of donor- and 4 ⁇ g of acceptor-expressing vectors using Lipofectamine 2000 (Invitrogen) in OptiMEM media for 24 hours at 37°C according to the manufacturer's instructions.
  • HEK293 cells were cultured as described for HeLa cells above and 1x10 s cells were seeded on MatTek 6 cm cell culture plates 24 h before transfection. Cells were transfected or double transfected as before and the OptiMem media was changed 6 h after transfection to DMEM with 10% FBS, 1% Pen/Strep, and 1% glutamate for 12 h before FRET measurements. EGF was added at a concentration of 0.05 ⁇ g/ml in 1.5 ml imaging buffer 4 minutes prior to FLIM experiments. mCitrine and mCherry were visualized with a 63x 1.25NA objective at a zoom factor of 4x. FLIM was performed on 5 fields per sample (4 minutes between FLIM readings).
  • Regions of interest were selected in ImageJ [44] and cell or membrane areas within were defined automatically using custom ImageJ macros. Fluorescent decay data were analyzed in IGOR Pro (WaveMetrics, Portland, Oregon) using the pFLIM software module [31] following the authors' instructions. The pFLIM module was also used for the generation of intensity, intensity-weighted lifetime and FRET efficiency images. Rafl / BRaf interaction
  • HeLa cells were cultured as described above. 2.5x105 cells were seeded on MatTek 6 cm cell culture plates 24 hours before transfection. Cells were transfected with 4 ⁇ g of donor only (control) or double transfected with 4 ⁇ g of donor- and 4 ⁇ g of acceptor-expressing vectors using Lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions; media was changed 6 hours after transfection to DMEM with 10% FBS, 1% Pen/Strep, and 1% glutamate for 12 h before FRET measurements. mCitrine lifetime was measured before and 2h after treatment with GDC-0879 lOuM or DMSO (as a control).
  • FLIM images were recorded with a 40x 1.25NA Oil PlanApo objective at a zoom factor of 1.7x (256x256 pixel). FLIM was performed on 3 fields per sample. Fluorescent decay data were analyzed with IGOR Pro / pFLIM as above.
  • HeLa cells were cultured at 37 and 5% C02 in DMEM (Gibco), supplemented with 10% FBS, 1% glutamate, and 1% pen/strep. 2.5x105 cells were seeded on MatTek 6 cm cell culture plates 24 hours before transfection. Cells were transfected with 4 ⁇ g of caspase3 sensor expressing vectors using Lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions; media was changed 6 hours after transfection to DMEM with 10% FBS, 1% Pen/Strep, and 1% glutamate for 12 h before FRET measurements. Media was changed to imaging buffer (DMEM without Phenol Red).
  • Staurosporine was added at a concentration of 2 ⁇ in 2 ml imaging buffer 10 min after ratiometric FRET experiment started. Images were acquired with a Leica TCS SP5 confocal microscope using a 63x 1.4 NA PL APO objective (Leica Microsystems GmbH). For each Caspase sensor, images were obtained every 5 minutes for 5 hours in 12 different positions of the same culture dish. Cells were excited with a 458 nm laser and donor and FRET channels were imaged with 2 hybrid detectors (HyD) with a 458/514 double dichroic beamsplitter and acquisition bandpass filters set between 469 and 507 nm and between 523 and 576 nm, respectively.
  • HyD hybrid detectors
  • Image size was 512x512 pixels and laser scanning speed was set to 400 Hz bidirectional. Laser AOTF and HyD voltage settings were kept constant for all the tested sensors for comparative purposes. Image processing and analysis was performed with FIJI software. Briefly, images were median filtered for noise reduction and background was subtracted from ROIs. After this processing ratio images were calculated dividing the FRET channel by the Donor channel. Ratiometric values of at least 30 cells were measured for each Caspase sensor.
  • rapamycin induces 100 % binding between FRB and FKBP12 domains (K D ⁇ 1 nM[14]). The donor emission with rapamycin thus becomes
  • the absolute FRET efficiency can be determined as described above at lower donor and acceptor concentrations (0.5 ⁇ ) where we do not observed peptide - domain interactions.
  • the unknown F0 is eliminated by combining equations 6 and 7 to F
  • FRET efficiency ⁇ E FRET efficiency based on donor quenching measured in vitro (plate reader); b E based on acceptor sensitized emission measured in vitro (plate reader); c E based on decrease of amplitude- weigh ted average lifetime (single measurement); d E based on decrease of photon count during same experiment (single measurement); Note, IDs refer to the full list of protein constructs given in a separate table.
  • 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 7 Overview amino acid and nucleotide sequences used in this study.
  • Non-limiting examples of reference and engineered polypeptides are provided.
  • Nucleotide sequences of synthetic protein constructs can be easily derived by combining the ucleotide sequences as provided in SEQ ID NOs: 41-68, 81, 92-97.
  • SH3 Domain Interactome Predicts Spatiotemporal Dynamics of Endocytosis Proteins.

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Abstract

L'invention concerne le domaine des protéines fluorescentes et leurs applications polyvalentes dans le domaine de la biologie moléculaire. Plus spécifiquement, l'invention concerne des procédés de production de protéines fluorescentes génétiquement modifiées en vue de l'obtention d'un FRET de haute efficacité (Transfert d'Énergie par Résonance de type Förster). En particulier, ces procédés consistent à fusionner une protéine fluorescente donneuse et receveuse à un module d'interaction auxiliaire à faible affinité. De plus, des paires de telles protéines fluorescentes génétiquement modifiées sont divulguées, ainsi que leurs utilisations dans des applications à base de FRET.
PCT/EP2012/075743 2011-12-15 2012-12-17 Procédés de production de protéines fluorescentes génétiquement modifiées à des fins d'amélioration du fret, produits et leurs utilisations WO2013087922A1 (fr)

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