WO2024089297A1 - Nouvelles protéines fluorescentes et bioluminescentes et systèmes rapporteurs pour la mesure ratiométrique de la luminescence - Google Patents

Nouvelles protéines fluorescentes et bioluminescentes et systèmes rapporteurs pour la mesure ratiométrique de la luminescence Download PDF

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WO2024089297A1
WO2024089297A1 PCT/EP2023/080279 EP2023080279W WO2024089297A1 WO 2024089297 A1 WO2024089297 A1 WO 2024089297A1 EP 2023080279 W EP2023080279 W EP 2023080279W WO 2024089297 A1 WO2024089297 A1 WO 2024089297A1
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luciferase
bioluminescent
protein
amino acid
acid sequence
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Jörg Urban
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Urban Joerg
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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
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0069—Oxidoreductases (1.) acting on single donors with incorporation of molecular oxygen, i.e. oxygenases (1.13)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/66—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving luciferase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y113/00—Oxidoreductases acting on single donors with incorporation of molecular oxygen (oxygenases) (1.13)
    • C12Y113/12—Oxidoreductases acting on single donors with incorporation of molecular oxygen (oxygenases) (1.13) with incorporation of one atom of oxygen (internal monooxygenases or internal mixed function oxidases)(1.13.12)
    • 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
    • G01N33/531—Production of immunochemical test materials
    • G01N33/532—Production of labelled immunochemicals
    • G01N33/535—Production of labelled immunochemicals with enzyme label or co-enzymes, co-factors, enzyme inhibitors or enzyme substrates
    • 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

Definitions

  • the present invention relates to methods to obtain enhanced variants of fluorescent and bioluminescent proteins based on ratiometric measurement of luminescence.
  • Disclosed are furthermore engineered variants of a yellow fluorescent protein (YFP) derived from Aequoria victoria GFP, variants of Renilla luciferase (Rluc) with blue-shifted emission (Clue), and bioluminescent fusion proteins comprising an inventive yellow fluorescent protein, including variants comprising Rluc or a variant of Rluc with red-shifted emission as donor luciferase (Yluc).
  • YFP yellow fluorescent protein
  • Rluc Renilla luciferase
  • Celue blue-shifted emission
  • bioluminescent fusion proteins comprising an inventive yellow fluorescent protein, including variants comprising Rluc or a variant of Rluc with red-shifted emission as donor luciferase (Yluc).
  • the invention pertains to reporter systems for ratiometric measurement of luminescence including a novel dual-color luminescence (DCL) system and
  • Bioluminescent proteins such as luciferases and bioluminescent fusion proteins are frequently used as intracellular reporters for the detection and quantification of biological processes, allowing to collect data with high sensitivity and a good signal to noise ratio.
  • Luciferases produce light by catalyzing one or more steps in the conversion of a luminogenic substrate into a chemiphore and providing a proteinaceous environment necessary for efficient light emission by the chemiphore.
  • Commonly used are mainly insect luciferases (e.g. firefly and click beetle luciferase), which produce light in combination with D-luciferin or derivatives thereof, and marine luciferases, which utilize luminogenic substrates comprising an imidazopyrazinone (imidazo[1 ,2-a]pyrazin-3(7H)-one) moiety.
  • Marine luciferases include secreted luciferases (e.g.
  • Gaussia, Cypridina, and Metridia luciferase and intracellular luciferases, whose activity is regulated by binding of calcium ions to calcium-binding motifs in the luciferase (e.g. Aequorin, Clythin, and Obelin) or which are not directly regulated by calcium (e.g. Renilla luciferase).
  • calcium-binding motifs in the luciferase e.g. Aequorin, Clythin, and Obelin
  • Renilla luciferase e.g. Renilla luciferase
  • luciferase NanoLucTM which has been derived from a secreted luciferase (Oplophorus luciferase), but can be expressed intracellularly (Markova et al., (2015), Biochemistry (Mose) 80(6): 714- 732; Cevenini et a/., (2016), Bioluminescence: Fundamentals and Applications in Biotechnology, 3 (154): 3-17).
  • Bioluminescent fusion proteins comprise a luciferase and at least one acceptor for bioluminescence resonance energy transfer (BRET), typically a fluorescent protein.
  • BRET bioluminescence resonance energy transfer
  • energy is transferred non-radiatively from a chemiphore to an acceptor fluorophore and light is emitted at longer wavelengths compared to light emitted from the chemiphore.
  • complementary fragments that assemble post-translationally into a luciferase are used as reporter, allowing for instance to quantify protein abundance via luminescence intensity using a small peptide-tag or to detect an interaction between polypeptides connected to such complementary fragments.
  • bioluminescent proteins which produce light with different substrates, are frequently combined into dual reporter systems. Due to the different properties of the bioluminescent proteins and substrates involved, these systems are typically used for successive measurements, wherein the first bioluminescent protein is inactivated following measurement of light emission in presence of its substrate to allow for detection of light emission in presence of the substrate utilized by the second bioluminescent protein.
  • Measurements of absolute levels of intracellularly generated luminescence are hampered by a varying light output due to intrinsic features of bioluminescent proteins and changing intracellular levels of substrate and reaction products, which depend also on transport processes across the plasma membrane and the cellular defense against oxidative stress. They are thus sensitive to changes in experimental conditions. Furthermore, they require accurate determination of a reference using a different technique such as measuring cell density for comparison between samples. These problems can be overcome by measuring luminescence ratiometrically, i.e. by determining the ratio of emission in two spectral ranges, using a reporter system for ratiometric measurement of luminescence.
  • Reporter systems have been developed, which comprise two luciferases that emit spectrally distinct light with the same luminogenic substrate, typically luciferases originating from insects (Almond et al., (2003), Promega Notes 85: 11-14; Gonzalez-Grandio et al., (2021 ), ACS Synth Biol 10(10): 2763-2766).
  • DCL dual-color luminescence
  • BRET systems which comprise a luciferase and an acceptor for BRET and allow to detect, if proteins of interest are in close proximity.
  • sample cells which comprise luciferase und acceptor
  • reference cells which comprise only the luciferase
  • the first BRET system disclosed has been a combination of Renilla luciferase and a yellow fluorescent protein (YFP) derived from Aequoria victoria GFP (avGFP) (EP1 088 233 B9). Since then, many different combinations of luciferases, luminogenic substrates and acceptors have been described (Sun et al., (2016), International Journal of Molecular Sciences, 17(10); Yeh and Ai, (2019), Annu Rev Anal Chem (Palo Alto Calif), 12(1 ): 129- 150), including BRET systems comprising NanoLucTM as luciferase (Hall et al., (2012), ACS Chem Biol, 7(11): 1848-1857).
  • YFP yellow fluorescent protein
  • BRET systems which contain a marine luciferase, occur naturally in different organisms.
  • the best-known examples stem from the coelenterates Aequoria victoria, Clytia gregaria and Renilla reniformis (Titushin et al., (2011), Protein Cell, 2(12): 957-972).
  • natural BRET systems often display highly efficient energy transfer, indicating that luciferase and acceptor must be in a complex allowing for optimal spacing and orientation of donor chemiphore and acceptor fluorophore. Nonetheless, studies with the BRET system of C.
  • reniformis is limited, since they contain a calcium-dependent luciferase (aequorin, clytin) and/or an acceptor that is an obligate dimer (cgreGFP, rGFP) (Loening et al., (2007), J Mol Biol, 374(4): 1017-1028; Malikova et al., (2011 ), Biochemistry, 50(20): 4232-4241).
  • the marine luciferases Renilla luciferase (Rluc) and NanoLucTM (NIuc), are of particular interest for the generation of ratiometric reporter systems, since they are smaller than insect luciferases, can be expressed intracellularly, produce light in a process that is not regulated by calcium-binding to the luciferase and does not require ATP. Both luciferases produce blue-green light and can be combined with fluorescent proteins that emit light in the green to red range of the spectrum to generate bioluminescent fusion proteins and BRET systems.
  • Rluc is a cytoplasmic luciferase, which produces light with Clz and various artificial substrates comprising an imidazopyrazinone moiety.
  • Previous studies have provided data about its structure and function and have shown that the emission spectrum of Rluc can be modified by altering residues in the vicinity of its substrate binding pocket.
  • Data about the influence of a number of mutations on properties such as intensity or spectral properties of light emitted by the luciferase have been published including data concerning the following mutations relevant for the invention: A54P, A55T, A123S, C124A, W153F, W156F, 1163V, I166L, M185V, K189E, P220L, P220V, P220M and I223V.
  • NIuc is an unrelated luciferase that produces light with the artificial substrate furimazine, which offers advantages compared to Rluc such as a smaller size and higher brightness, but is apparently less suitable for the generation of spectral variants.
  • Fluorescent proteins contain in their mature form a fluorophore, which emits light following excitation via absorption of a photon or via non-radiative energy transfer (resonance energy transfer) from a donor chromophore or chemiphore.
  • a wide array of fluorescent proteins with different types of fluorophores have been developed from naturally occurring variants. The usefulness of fluorescent proteins as molecular tools depends on their spectral characteristics as well as additional parameters including protein folding efficiency at different temperatures, fluorophore maturation rate, photostability, sensitivity to external conditions, the possibility to modify their spectral properties reversibly or irreversibly and the oligomeric state of the molecule.
  • Fluorescent proteins have a common structure termed p-barrel in which a central a-helix is surrounded by 11 p-sheets.
  • the fluorophore is formed autocatalytically from residues in the a-helix in a process called maturation.
  • Folding and maturation require the presence of a core folding unit encompassing almost the entire P-barrel structure (amino acids 2 to 230 of avGFP), which in avGFP is followed by an unstructured C-terminal peptide without importance for functionality (amino acids 231 to 238). It is possible to insert other polypeptides at certain points of the core folding unit without loss of functionality (US 6,469,154 B1 ).
  • functional fluorescent proteins can be assembled post- translationally from complementary fragments (EP 0 966 685 B9). It is also possible to generate a circular permuted variant, wherein complementary fragments derived from a fluorescent protein are comprised in the same polypeptide in an altered order (US 6,699,687 B1 ). Another technique known in the art is to create variants capable of folding in an oxidizing environment by altering certain residues in a fluorescent protein that can be oxidized (Costantini et al., (2015), Nat Commun, 6: 7670). It is also possible to alter multiple residues in a fluorescent protein without substantial loss of functionality (US 6,699,687 B1 ). Several variants with altered spectral properties (e.g.
  • BFP, CFP, GFP, GFP2, and YFP have been derived from avGFP, whose spectral characteristics are defined by mutations within or close to the fluorophore and which typically contain additional mutations for optimization of the fluorophore environment (Shaner et a/., (2007), Cell Sci, 120(Pt 24): 4247-4260).
  • YFP variants derived from avGFP have been created by introducing a tyrosine residue in position 203 (T203Y), whose n-stacking interaction with the p-hydroxy-phenyl moiety of the fluorophore causes red-shifted emission (Ormo et a/., (1996), Science, 273(5280): 1392- 1395). Further development led to variants with enhanced properties including YFP10B, YFP10C, Topaz, Citrine, mVenus, YPet, SYFP2, sfYFP, FFTS-YFP, Citrine2, and Achilles.
  • fluorescent proteins with special properties have been developed from YFP variants derived from avGFP including Reach, sReach, Dreiklang, moxYFP, ShadowY, and SPOON (www.fpbase.org).
  • Some of these variants contain mutations relevant for this invention: S65G, S72A, T203Y (Ormo et a/., (1996), Science, 273(5280): 1392-1395); F46L, M153T, V163A, S175G (Nagai et a/., (2002), Nat Biotechnol, 20(1 ): 87-90); S30R, Y39I, F99S, N105K, Y145F, S175G (Ottmann et a/., (2009), Biol Chem, 390(1 ): 81-90).
  • YFP variants derived from avGFP such as mVenus and Citrine are well suited as BRET acceptors for use with Rluc or NIuc, since their emission with a maximum at about 529 nm is better spectrally separated from the emission of the luciferase than those of GFP variants. Compared to fluorescent proteins with further red-shifted emission, YFP variants are advantageous due to higher brightness and shorter maturation time.
  • BRET efficiency of BRET systems and bioluminescent fusion proteins which have not been derived from a naturally occurring BRET system with interacting components, is limited due to a random orientation of luciferase and acceptor and in some cases due to low BRET efficiency or slow fluorophore maturation of the fluorescent protein used as acceptor.
  • Methods to optimize fluorescent or bioluminescent proteins typically rely on purification and physical characterization of variants of interest. Thereby, the number of variants that can be processed is limited due to laborious procedures and the resulting data are not necessarily indicative that the analyzed protein is suitable for use as reporter or component of a reporter system in cellula.
  • Versions of the inventive method comprising BRET measurements with additional test fusion proteins and/or measurements of relative fluorescence intensity in cellula allow to compare multiple properties of fluorescent proteins without purification, alleviating the development of improved variants of a fluorescent protein via combination of multiple mutations.
  • incorporación of such a YFP variant allowed to create bioluminescent fusion proteins with enhanced BRET efficiency.
  • Rluc and Yluc-1 when co-expressed in a cell, emit spectrally well separated light in presence of Clz at a ratio that remains relatively constant over a certain time.
  • DCL dual-color luminescence
  • ratiometric measurements allowed to identify mutations in Rluc and in the luciferase comprised in Yluc-1 (or YR), which are suitable to introduce or enhance certain desired properties such as a better spectral separation between light produced by the bioluminescent proteins, a higher emission by one bioluminescent protein relative to emission by the other, or an emission with a relatively constant ratio over a certain time by both bioluminescent proteins in cellula.
  • Clue and Yluc variants when co-expressed in a cell, emit light in presence of Clz at a relatively constant ratio that is spectrally better separated than light emitted by cells comprising Rluc and Yluc-1 , providing an advanced dual-color luminescence system.
  • BRET systems which yield higher BRET ratios compared to BRET systems comprising a commonly used YFP variant derived from avGFP as acceptor.
  • the invention thus provides:
  • a method comprising the determination of a BRET ratio and a color value via measurement of light emission in three different spectral ranges in cellula from a luciferase and a test fusion protein comprising the same luciferase and a fluorescent protein.
  • a yellow fluorescent protein with an amino acid sequence derived from SEQ ID NO: 1 containing mutation T203Y which in addition comprises at least two of the following mutations: L42F, T43K, F46V, F64V, F84Y, 1123V, E142A, R168K, Y182V, Q204V, Q204R, A206R, L207V, L207I, S208Y, V219A, L220V, L220I, L221 I, V224M, and V224I.
  • the inventive yellow fluorescent protein further comprises at least one of the following mutations: S30R, Y39I, F46L, S65G, S72A, F99S, N105K, Y145F, M153T, V163A, and S175G.
  • the inventive yellow fluorescent protein comprises an amino acid sequence with at least 80 % identity to any one of SEQ ID NOs: 23 or 25.
  • the inventive yellow fluorescent protein exhibits in cellula in presence of Clz enhanced BRET efficiency compared to SYFP2 in a test fusion protein based on SEQ ID NO: 3 (BEYFP -Rluc:SYFP2-Rluc > 1 ) ⁇
  • the inventive yellow fluorescent protein exhibits in cellula in presence of Clz strongly enhanced BRET efficiency compared to SYFP2 in a test fusion protein based on SEQ ID NO: 3 (BEYFP-RIUC:SYFP2-RIUC > 3), which can be attributed in part to an interaction between the yellow fluorescent protein and Rluc.
  • amino acid sequence of the inventive yellow fluorescent protein comprises SEQ ID NO: 23, 24, 25, 53, 54 or 55.
  • the inventive luciferase further comprises at least one of the following mutations: A55T, A123S, D154E, E155D, I166L, and M185V.
  • the amino acid sequence of the inventive luciferase has at least 80 % identity to SEQ ID NO: 34.
  • the inventive luciferase emits in cellula in presence of Clz less light in the spectral range between 520 and 550 nm relative to emission in the spectral range between 430 and 490 nm compared to a protein with amino acid sequence SEQ ID NO: 18.
  • the inventive luciferase has amino acid sequence SEQ ID NO: 34, 49, 102 or 103.
  • a bioluminescent fusion protein which comprises the yellow fluorescent protein described in [2] and a luciferase.
  • the luciferase comprised in the inventive bioluminescent fusion protein produces light with a luminogenic substrate that comprises an imidazo[1 ,2-a]pyrazin-3(7H)-one moiety.
  • the amino acid sequence of the luciferase comprised in the inventive bioluminescent fusion protein has at least 80 % identity to any one of SEQ ID NOs: 18 or 19.
  • the yellow fluorescent protein comprised in the inventive bioluminescent fusion protein exhibits in cellula in presence of a luminogenic substrate (suitable to produce light with the luciferase comprised in the inventive bioluminescent fusion protein) strongly enhanced BRET efficiency compared to SYFP2 in a test fusion protein based on SEQ ID NO: 3 containing the luciferase comprised in the inventive bioluminescent fusion protein, which can be attributed in part to an interaction between yellow fluorescent protein and luciferase.
  • a luminogenic substrate suitable to produce light with the luciferase comprised in the inventive bioluminescent fusion protein
  • the luciferase comprised in the bioluminescent fusion protein of [5] further comprises at least one of the following mutations: A123S, C124A, W153F, D154E, E155D, I166L, and L284Y.
  • the luciferase comprised in the bioluminescent fusion protein of [5] has an amino acid sequence with at least 80 % identity to SEQ ID NO: 37.
  • the inventive bioluminescent fusion protein of [5] emits in cellula in presence of Clz more light in the spectral range between 520 and 550 nm relative to emission in the spectral range between 430 and 490 nm compared to a protein with amino acid sequence SEQ ID NO: 20.
  • the luciferase comprised in the bioluminescent fusion protein of [5] has amino acid sequence SEQ ID NO: 37 or 38.
  • a dual-color luminescence system comprising two bioluminescent proteins that produce spectrally distinct light with the same luminogenic substrate, wherein the first bioluminescent protein comprises a luciferase that can be expressed intracellularly and whose activity is not regulated by binding of calcium ions to calcium-binding motifs in the luciferase; the second bioluminescent protein comprises a bioluminescent fusion protein comprising a luciferase and at least one fluorescent protein; the amino acid sequence of the luciferase comprised in the second bioluminescent protein has at least 60 % identity, preferably at least 80 % identity to the amino acid sequence of the luciferase comprised in the first bioluminescent protein; and the luminogenic substrate comprises an imidazo[1 ,2- a]pyrazin-3(7H)-one moiety.
  • the second bioluminescent protein of the inventive dual-color luminescence system comprises the yellow fluorescent protein described in [2], Preferably, the second bioluminescent protein of the inventive dual-color luminescence system emits in cellula in presence of the luminogenic substrate at least 30-fold more light in the spectral range between 520 and 550 nm relative to emission in the spectral range between 430 and 490 nm compared to the first bioluminescent protein of the inventive dualcolor luminescence system.
  • the luciferase comprised in the second bioluminescent protein of the inventive dual-color luminescence system has an amino acid sequence with at least 80 % identity to any of the SEQ ID NOs: 18 or 19.
  • the luciferase comprised in the first bioluminescent protein of the inventive dualcolor luminescence system is the luciferase described in [3].
  • the bioluminescent fusion protein comprised in the second bioluminescent protein of the inventive dual-color luminescence system is the bioluminescent fusion protein described in [5].
  • a BRET system comprising a luciferase and an acceptor for BRET, wherein the acceptor comprises the yellow fluorescent protein described in [2].
  • the luciferase comprised in the inventive BRET system produces light with a luminogenic substrate that comprises an imidazo[1 ,2-a]pyrazin-3(7H)-one moiety.
  • the luciferase comprised in the inventive BRET system has an amino acid sequence with at least 80 % identity to any one of SEQ ID NOs: 18 or 19 or is the luciferase described in [3].
  • the luciferase comprised in the inventive BRET system has amino acid sequence SEQ ID NO: 18, 19, 34, 46, 49, 102 or 103.
  • the inventive BRET system is a BRET system with interacting components, wherein an inventive yellow fluorescent protein comprised in the acceptor exhibits in cellula in presence of a luminogenic substrate (suitable to produce light with the luciferase comprised in the inventive BRET system) strongly enhanced BRET efficiency compared to SYFP2 in a test fusion protein based on SEQ ID NO: 3 containing the luciferase comprised in the inventive BRET system, which can be attributed in part to an interaction between yellow fluorescent protein and luciferase.
  • a luminogenic substrate suitable to produce light with the luciferase comprised in the inventive BRET system
  • a cell comprising any of the proteins described in [2] to [5], the first and the second bioluminescent protein of the dual-color luminescence system described in [6], the luciferase and the acceptor comprised in the BRET system described in [7], or the nucleic acid or combination of nucleic acids described in [8].
  • a method comprising the determination of an emission ratio via measurement of light emission in presence of the same luminogenic substrate in two different spectral ranges from a cell comprising the first bioluminescent protein, a cell comprising the second bioluminescent protein, and a cell comprising both the first and the second bioluminescent protein of a dual-color luminescence system as described in [6].
  • the inventive method of [10] is a 'method for conducting ratiometric measurements of luminescence using a dual-color luminescence system', which comprises:
  • sample cells comprising one of the bioluminescent proteins in a way that its emission yields information about a biological process of interest and the other bioluminescent protein in a way that it provides an internal reference or that its emission yields information about the same or a different biological process of interest.
  • the BRET efficiency of a yellow fluorescent protein of interest compared to SYFP2 in a test fusion protein based on SEQ ID NO: 3 comprising the luciferase luc in cellula in presence of a luminogenic substrate is to be determined using the following method:
  • Cassettes for expression of 3FLAG-Rluc and SYFP2-Rluc shall have nucleotide sequence SEQ ID NO: 29 and 30, respectively.
  • Cassettes for expression of YFP-Rluc shall have a nucleotide sequence derived from SEQ ID NO: 30, wherein SEQ ID NO: 31 encoding amino acids 2-230 of SYFP2 has been replaced by a nucleotide sequence encoding the corresponding amino acid sequence (based on an alignment with SYFP2) of the yellow fluorescent protein of interest.
  • Cassettes for expression of 3FLAG-luc, SYFP2-luc, and YFP-luc containing a luciferase other than Rluc shall have a nucleotide sequence derived from the expression cassettes described above for 3FLAG-Rluc, SYFP2-Rluc, and YFP-Rluc, respectively, wherein SEQ ID NO: 32 encoding Rluc has been replaced by a nucleotide sequence encoding the luciferase.
  • Light emission by a bioluminescent protein in cellula in presence of a luminogenic substrate in the spectral range between 520 and 550 nm relative to emission in the spectral range between 430 and 490 nm is to be determined using the following method: Growth of a culture of cells (S. cerevisiae) containing an expression cassette allowing for expression of the bioluminescent protein at 30 °C in SC-Q medium as described above to mid-log phase. Addition of the luminogenic substrate, incubation at 30 °C and measurement using filters 460/60 and 535/30 as described above. Calculation of the color ratio (535:460) for the bioluminescent protein.
  • Figure 1 Dependence of light emission by Rluc in cellula on experimental conditions.
  • a Time course experiment showing light emission from cultures of cells expressing Rluc with different density. Cultures were inoculated at a ratio of 1 :2:4:8:16 and grown for 6 hours prior to addition of medium containing Clz.
  • B and C Light emission by Rluc in presence of different concentrations of Tween 20 or ascorbic acid added 10 min prior to addition of Clz.
  • D Light emission by cultures of cells not expressing Rluc (wt) in presence of Clz. Cells expressing Rluc were added after 30 min.
  • the horizontal axis in A to D indicates the time after mixing of Clz and medium, with measurements starting ca. 3 min after mixing.
  • Rluc was expressed using expression cassette Clucx. In A to C Clz was used at a final concentration of 5 pM.
  • Figure 4 Luminescence spectra obtained with cultures of cells expressing Cluc-7, Rluc, or Yluc-9 in low-fluorescence medium containing 5 pM Clz. Values have been normalized to the emission maximum. Constancy of emission ratios over time. A Time course following addition of Clz determined with cells expressing bioluminescent proteins individually and in combination. B Luminescence detected using filter 460/60 in the same experiment for some individually expressed bioluminescent proteins. C Influence of Clz concentration on the emission ratio of cells co-expressing Cluc-7 and Yluc-9. The horizontal axis in A to C shows the time after mixing of Clz and medium.
  • Cluc-7 was expressed from a genomic locus (gen) or from episomal plasmid pRS 416 (epi) and Yluc-9 was expressed from a different genomic locus. Emission ratios were determined 3 days and 34 days after transformation of Yluc-9 expressing cells with the Cluc-7 expression cassette for 45 cultures each, inoculated with different colonies from the same transformation. Emission ratios were calculated relative to the average emission ratio determined for cultures of cells expressing both bioluminescent proteins from a genomic locus for the respective day. : DCL analysis of protein degradation induced by commonly used tags.
  • Emission ratios were determined in strains expressing Yluc-9 as internal reference and calculated for N- or C-terminally tagged Cluc-7 relative to untagged Cluc-7. Shown are relative emission ratios recorded following addition of cycloheximide CHX at a final concentration of 100 pg/ml, depicted at a logarithmic scale. Tabular data show steady-state relative emission ratios recorded prior to addition of CHX.
  • Figure 9 BRET analysis of the formation of a complex consisting of FRB-Rluc, FKBP12- YFP and rapamycin (rap). A Comparison of BRET values obtained with different YFP variants as acceptor following incubation with drug vehicle or rap for 60 min at 33 °C in presence of 5
  • FIG. 10 BRET analysis of the interaction between modified components of protein kinase A.
  • Figure 11 BRET analysis of the interaction between Rluc-Ras2 and Raf RB D-YFP B using mutant variants of Ras2 that interact similar to GDP-loaded Ras2 (Ras2 T42N ) or contain higher levels of GTP (Ras2 G19V ). Clz was added at a concentration of 5 jiM.
  • Figure 12 Characterization of Rluc-B in comparison with Rluc. Time course experiment showing emission ratios recorded with cells co-expressing Rluc or Rluc-B with the corresponding fusion protein YR or YR-B.
  • the invention provides the method described in [1],
  • the inventive method is a 'method to obtain enhanced fluorescent proteins using measurements of BRET efficiency in test fusion proteins and of relative fluorescence intensity', which comprises:
  • RECTIFIED SHEET (RULE 91) ISA/EP [a] A cell comprising a test fusion protein comprising one of the luciferases L1 or L2 and a fluorescent protein F (sample) and a cell comprising the same luciferase without acceptor for BRET (ref).
  • test fusion protein comprises a degron.
  • a cell comprising a first polypeptide comprising F and a second polypeptide comprising a fluorescent protein F2, wherein F2 has distinct spectral characteristics compared to F and is suitable to provide a reference for measurements of brightness of F in cellula.
  • [h] Alteration of the amino acid sequence of F in a test fusion protein comprised in a cell as in [a] or [b] and determination of BR and CV values as in [d] and [e] in comparison with a cell as in [a] or [b] that comprises a different fluorescent protein with similar spectral properties instead of F in an otherwise identical test fusion protein.
  • [i] Alteration of the amino acid sequence of F in a polypeptide comprised in a cell as in [c] and analysis of intracellular fluorescence as in [f] and [g] in comparison with a cell as in [c], which comprises a different fluorescent protein with similar spectral properties instead of F in an otherwise identical polypeptide.
  • BRET measurements are conducted with cells of S. cerevisiae grown in a medium that produces little autoluminescence in presence of the luminogenic substrates used to generate light with L1 and L2, which express the polypeptides of [a] or [b] under control of a strong constitutive promoter from an episomal or genomic locus, using devices capable to detect light emission within different spectral ranges of interest such as a microplate reader equipped with filters with corresponding windows of transparency.
  • amino acid sequences of luciferases L1 and L2 have less than 60 % identity.
  • measurements of fluorescence are conducted in cells of S. cerevisiae grown in a medium with low background fluorescence, which contain one or more cassettes for co-expression of the polypeptides of [c] under control of a strong constitutive promoter from a genomic locus, using devices suitable to excite fluorophores and measure their emission within different spectral ranges such as a fluorescence microscope.
  • cells as in [a] or [b], which comprise in addition a polypeptide comprising F2 are used to conduct bioluminescence measurements as in [d] and [e], measurements of fluorescence intensity as in [f] and [g], and the generation and analysis of altered variants of F as in [h] and [i].
  • the inventive method is used to compare properties of fluorescent proteins such as BRET efficiency in a fusion protein comprising the fluorescent protein and a luciferase, presence of an affinity between the fluorescent protein and a luciferase causing enhanced BRET efficiency in a fusion protein comprising them, relative position of peak emission wavelength, fluorophore maturation rate, intracellular brightness, and photostability.
  • fluorescent proteins such as BRET efficiency in a fusion protein comprising the fluorescent protein and a luciferase
  • presence of an affinity between the fluorescent protein and a luciferase causing enhanced BRET efficiency in a fusion protein comprising them, relative position of peak emission wavelength, fluorophore maturation rate, intracellular brightness, and photostability.
  • the inventive method is used to compare properties of closely related fluorescent proteins containing different mutations, which contain an unstructured C-terminal peptide with the amino acid sequence of avGFP (amino acids 231 -238).
  • the inventive method is used to compare properties of unrelated fluorescent proteins with similar spectral properties, which contain an unstructured C-terminal peptide with the amino acid sequence of avGFP (amino acids 231- 238).
  • the inventive method is used to identify mutations or combinations of mutations in a fluorescent protein, which entail a desired property. This includes validation of the usefulness of mutations already known to entail a desired property in combination with other mutations.
  • the inventive method is used to create variants of a fluorescent protein with enhanced properties.
  • the inventive method is used to create a fluorescent protein, which yields enhanced BRET ratios specifically in combination with a particular luciferase that can be attributed to an affinity between the fluorescent protein and the luciferase.
  • the presence of a physical interaction between a luciferase and a fluorescent protein causing enhanced BRET efficiency can be concluded based on evidence that in cellula a high BRET efficiency of a fusion protein comprising the luciferase and the fluorescent protein is strongly reduced in a similar fusion protein, wherein the luciferase has been replaced by an unrelated luciferase; is strongly reduced for fusion proteins containing luciferase and fluorescent protein in a different order and therefore presumably subject to sterical inhibition; depends on the presence of one or a few amino acids, whose side chains are exposed at the surface of the luciferase and/or the fluorescent protein and are located within a surface area of the luciferase or the fluorescent protein that could function as interface for interaction with
  • the invention provides the yellow fluorescent protein described in [2],
  • the inventive yellow fluorescent protein has amino acid sequence SEQ ID NO: 15 (YFP B ) corresponding to avGFP (S30R, Y39I, F46V, F64V, S65G, S72A, F84Y, F99S, N105K, E142A, Y145F, M153T, V163A, S175G, T203Y, A206R, L207V, S208Y, V219A, L220V, L221 I, V224M).
  • avGFP S30R, Y39I, F46V, F64V, S65G, S72A, F84Y, F99S, N105K, E142A, Y145F, M153T, V163A, S175G, T203Y, A206R, L207V, S208Y, V219A, L220V, L221 I, V224M.
  • the inventive yellow fluorescent protein has amino acid sequence SEQ ID NO: 16 (YFP B N ) corresponding to avGFP (S30R, Y39I, F46V, F64V, S65G, S72A, F84Y, F99S, N105K, E142A, Y145F, M153T, V163A, S175G, T203Y, Q204R, L207V, S208Y, V219A, L220V, L221 I, V224M).
  • avGFP S30R, Y39I, F46V, F64V, S65G, S72A, F84Y, F99S, N105K, E142A, Y145F, M153T, V163A, S175G, T203Y, Q204R, L207V, S208Y, V219A, L220V, L221 I, V224M.
  • the inventive yellow fluorescent protein has amino acid sequence SEQ ID NO: 50 (YFP B D ) corresponding to avGFP (S30R, Y39I, F46V, F64V, S65G, S72A, F84Y, F99S, N105K, E142A, Y145F, M153T, V163A, S175G, T203Y, L207V, S208Y, V219A, L220V, L221 I, V224M).
  • avGFP S30R, Y39I, F46V, F64V, S65G, S72A, F84Y, F99S, N105K, E142A, Y145F, M153T, V163A, S175G, T203Y, L207V, S208Y, V219A, L220V, L221 I, V224M.
  • the inventive yellow fluorescent protein has amino acid sequence SEQ ID NO: 17 (YFP A ) corresponding to avGFP (S30R, Y39I, L42F, F46L, F64V, S65G, S72A, F84Y, F99S, N105K, 1123V, E142A, Y145F, M153T, R168K, S175G, Y182V, T203Y, A206R, L207I, S208Y, V219A, L220I, L221 I, V224I).
  • avGFP S30R, Y39I, L42F, F46L, F64V, S65G, S72A, F84Y, F99S, N105K, 1123V, E142A, Y145F, M153T, R168K, S175G, Y182V, T203Y, A206R, L207I, S208Y, V219A, L220I, L221 I, V224I).
  • the inventive yellow fluorescent protein has amino acid sequence SEQ ID NO: 51 (YFP A ' N ) corresponding to avGFP (S30R, Y39I, L42F, F46L, F64V, S65G, S72A, F84Y, F99S, N105K, 1123V, E142A, Y145F, M153T, R168K, S175G, Y182V, T203Y, Q204R, L207I, S208Y, V219A, L220I, L221 I, V224I).
  • avGFP S30R, Y39I, L42F, F46L, F64V, S65G, S72A, F84Y, F99S, N105K, 1123V, E142A, Y145F, M153T, R168K, S175G, Y182V, T203Y, Q204R, L207I, S208Y, V219A, L220I, L221 I, V224
  • the inventive yellow fluorescent protein has amino acid sequence SEQ ID NO: 52 (YFP A ' D ) corresponding to avGFP (S30R, Y39I, L42F, F46L, F64V, S65G, S72A, F84Y, F99S, N105K, 1123V, E142A, Y145F, M153T, S175G, Y182V, T203Y, L207I, S208Y, V219A, L220I, L221 I, V224I).
  • avGFP S30R, Y39I, L42F, F46L, F64V, S65G, S72A, F84Y, F99S, N105K, 1123V, E142A, Y145F, M153T, S175G, Y182V, T203Y, L207I, S208Y, V219A, L220I, L221 I, V224I).
  • the inventive yellow fluorescent protein has amino acid sequence SEQ ID NO: 23, 24, 53, 25, 54 or 55.
  • the invention includes a yellow fluorescent protein, which
  • the yellow fluorescent protein according to the invention is connected to one or more polypeptides such as polypeptides of interest, linkers, degrons, targeting sequences, or tags.
  • polypeptides of interest such as polypeptides of interest, linkers, degrons, targeting sequences, or tags.
  • linkers such as polypeptides of interest, linkers, degrons, targeting sequences, or tags.
  • the invention provides the luciferase described in [3].
  • the luciferase has amino acid sequence SEQ ID NO: 34 (Cluc- 9) corresponding to Rluc (A54P, A55T, A123S, 1140V, W153F, D154E, E155D, I166L, M185V, S218T, P220L, L225I).
  • the luciferase has amino acid sequence SEQ ID NO: 102 (Cluc-10) corresponding to Rluc (A54P, A55T, A123S, 1140V, W153F, D154E, E155D, W156F, I166L, M185V, S218T, P220L, L225I).
  • the luciferase has amino acid sequence SEQ ID NO: 49 (Cluc-11) corresponding to Rluc (A54P, A55T, A123S, 1140V, W153F, D154E, E155D, I166L, S218T, P220L, L225I).
  • the luciferase has amino acid sequence SEQ ID NO: 103 (Cluc-12) corresponding to Rluc (A54P, A55T, A123S, 1140V, W153F, D154E, E155D, W156F, I166L, S218T, P220L, L225I).
  • the invention includes a bioluminescent protein, which
  • the luciferase according to the invention is connected to one or more polypeptides such as polypeptides of interest, linkers, degrons, targeting sequences, or tags.
  • the invention provides the bioluminescent fusion protein described in [4],
  • inventive bioluminescent fusion protein comprises the yellow fluorescent protein described in [2], Preferred and particular embodiments of the inventive yellow fluorescent protein are described above in relation to aspect [2] and are fully applicable to the inventive bioluminescent fusion protein.
  • inventive bioluminescent fusion protein comprises an inventive yellow fluorescent protein with an amino acid sequence comprising any of the SEQ ID NOs: 23, 24, 25, 53, 54 or 55.
  • the inventive bioluminescent fusion protein comprises a luciferase with amino acid sequence SEQ ID NO: 18 or with an amino acid sequence that has at least 80 % identity to SEQ ID NO: 18.
  • the inventive bioluminescent fusion protein comprises a luciferase with amino acid sequence SEQ ID NO: 19 or with an amino acid sequence that has at least 80 % identity to SEQ ID NO: 19.
  • the inventive bioluminescent fusion protein comprises one or more additional acceptors for BRET such as an additional copy of the inventive yellow fluorescent protein.
  • the invention includes a bioluminescent fusion protein, which is assembled post-translationally from complementary fragments derived from the bioluminescent fusion protein described in [4].
  • the bioluminescent fusion protein according to the invention is connected to one or more polypeptides such as polypeptides of interest, linkers, degrons, targeting sequences, or tags.
  • bioluminescent fusion protein described in [4] is the bioluminescent fusion protein described in [5].
  • the bioluminescent fusion protein described in [5] comprises a yellow fluorescent protein with amino acid sequence SEQ ID NO: 23 and a luciferase with amino acid sequence SEQ ID NO: 37 (Rluc-Y12) corresponding to Rluc (G118I, A123S, C124A, A126G, D154E, E155D, 1163V, I166L, I223V, L225I, L284Y), ideally has amino acid sequence SEQ ID NO: 41 (Yluc-12).
  • the bioluminescent fusion protein described in [5] comprises a yellow fluorescent protein with amino acid sequence SEQ ID NO: 23 and a luciferase with amino acid sequence SEQ ID NO: 38 (Rluc-Y13) corresponding to Rluc (G118I, A123S, C124A, A126G, W153F, D154E, E155D, 1163V, I166L, K189E, I223V, L225I, L284Y), ideally has amino acid sequence SEQ ID NO: 42 (Yluc-13).
  • the invention includes a bioluminescent fusion protein, which
  • the invention provides the dual-color luminescence (DCL) system described in [6].
  • the invention includes a DCL system, wherein the first and/or the second bioluminescent protein of the DCL system is assembled post-translationally from complementary fragments.
  • the first bioluminescent protein and/or the second bioluminescent protein comprised in a DCL system according to the invention is connected to one or more polypeptides such as polypeptides of interest, linkers, degrons, targeting sequences, or tags.
  • the first bioluminescent protein of the inventive DCL system comprises the inventive luciferase described in [3], ideally a luciferase with amino acid sequence SEQ ID NO: 34 or 102
  • the second bioluminescent protein of the inventive DCL system comprises the inventive bioluminescent fusion protein described in [5], ideally a bioluminescent fusion protein with amino acid sequence SEQ ID NO: 41 or 42.
  • inventive proteins described above in relation to aspect [3] and [5], respectively are fully applicable to an inventive DCL system comprising any of the respective inventive proteins.
  • the invention provides the BRET system described in [7],
  • the inventive BRET system comprises the inventive yellow fluorescent protein.
  • Preferred and particular embodiments of the inventive yellow fluorescent protein are described above in relation to aspect [2] and are fully applicable to the inventive BRET system.
  • the inventive BRET system comprises a luciferase with amino acid sequence SEQ ID NO: 18, 34, 46, 49, 102 or 103 and an acceptor for BRET with an amino acid sequence comprising SEQ ID NO: 23.
  • the inventive BRET system comprises a luciferase with amino acid sequence SEQ ID NO: 18, 34, 46, 49, 102 or 103 and an acceptor for BRET with an amino acid sequence comprising SEQ ID NO: 24.
  • the inventive BRET system comprises a luciferase with amino acid sequence SEQ ID NO: 19 and an acceptor for BRET with an amino acid sequence comprising SEQ ID NO: 24.
  • luciferase and acceptor comprised in the inventive BRET system are comprised within one polypeptide or comprised in two or more polypeptides.
  • luciferase and/or acceptor comprised in a BRET system according to the invention are connected to one or more polypeptides such as polypeptides of interest, linkers, degrons, targeting sequences, or tags.
  • polypeptides such as polypeptides of interest, linkers, degrons, targeting sequences, or tags.
  • This may include for instance polypeptides that are subject to posttranslational modification or that provide affinity for molecules such as polypeptides, nucleic acids, lipids, sugar compounds, metabolites, or ligands.
  • the inventive BRET system is a BRET system with interacting components.
  • the inventive BRET system with interacting components is used to analyze a biological process by detecting, if a luciferase and an acceptor for BRET are brought into proximity by additional factors whose interaction, abundance or localization depends on the biological process of interest.
  • the inventive BRET system with interacting components is used to analyze a biological process by detecting, if an interaction between a luciferase and an acceptor for BRET is affected by another polypeptide with affinity for components of the inventive BRET system, wherein interaction, abundance, or localization of luciferase, acceptor and/or polypeptide with affinity for components of the inventive BRET system depends on the biological process of interest.
  • the inventive BRET system with interacting components is used to detect interactions between cellular components.
  • the inventive BRET system with interacting components is used to detect binding of a biological or non-biological molecule to a cellular component.
  • the inventive BRET system with interacting components is used to quantify the abundance of a cellular component.
  • the inventive BRET system with interacting components is used to detect a modification of a cellular component.
  • the inventive BRET system with interacting components is used to analyze the localization of a cellular component.
  • the inventive BRET system with interacting components is used to analyze environmental conditions based on the principle that the functioning of a modified acceptor is sensitive to changes of said environmental conditions. Possible modifications include, but are not limited to, insertion of polypeptides into normal or circular permuted versions of the acceptor or introduction of residues that can be oxidized. Environmental conditions that could be sensed include, but are not limited to, temperature, pH, redox state, and the concentration of ions.
  • the invention provides the nucleic acid or combination of nucleic acids described in [8].
  • Preferred and particular embodiments described above in relation to aspects [1] to [5], respectively, are fully applicable to the inventive nucleic acids or combinations of nucleic acids.
  • the invention provides the cell described in [9]. Preferred and particular embodiments described above in relation to aspects [1] to [8], respectively, are fully applicable to the inventive cell.
  • the invention includes the method described in [10].
  • the method is used to generate a bioluminescent protein with certain properties desired e.g. for use as component of a dual-color luminescence or BRET system, such as a better spectral separation between light produced by the bioluminescent protein and light produced by a second bioluminescent protein, a higher emission by the bioluminescent protein relative to emission by the second bioluminescent protein in cells co-expressing both proteins, or an emission with a relatively constant ratio over a certain time by both bioluminescent proteins in cells co-expressing both proteins.
  • certain properties desired e.g. for use as component of a dual-color luminescence or BRET system, such as a better spectral separation between light produced by the bioluminescent protein and light produced by a second bioluminescent protein, a higher emission by the bioluminescent protein relative to emission by the second bioluminescent protein in cells co-expressing both proteins, or an emission with a relatively constant ratio over a certain time by both bioluminescent proteins
  • the inventive method is the 'method to conduct ratiometric measurements of luminescence using a dual-color luminescence system' described in [10].
  • the method includes a cell comprising an expression cassette allowing for expression of a polypeptide comprising a component of the inventive DCL system, or a cell comprising two or more expression cassettes allowing for expression of polypeptides, which assemble post-translationally into a protein complex comprising a component of the inventive DCL system, as part of episomal or genomic DNA, ideally as part of genomic DNA.
  • the expression cassette(s) may allow for expression of a polypeptide or protein complex comprising a component of the inventive DCL system and one or more additional polypeptides such as polypeptides of interest, linkers, degrons, targeting sequences, or tags.
  • the expression cassette(s) may contain genetic elements that affect the transcription of DNA into mRNA or the translation of mRNA into polypeptides by altering processes such as transcriptional or translational initiation, elongation, or termination, or the processing, transport, storage, or stability of mRNA. This may be due to the presence of one or more nucleic acid sequences or sequence motifs with biological activity or the composition of the nucleic acid sequence (e.g. presence of synonymous codons in a nucleic acid encoding a polypeptide).
  • cells or tissues comprising a cell as described in [10] item [a] to [d] are grown and light emission in presence of a luminogenic substrate is measured in a suitable growth medium, ideally a transparent and essentially colorless medium that produces little autoluminescence in presence of the luminogenic substrate.
  • measurement of light emission as described in [10] item [e] is done concomitantly or successively in a short period of time, using devices suitable to detect luminescence within different spectral ranges such as a microplate reader or an imaging device equipped with filters with corresponding windows of transparency.
  • polypeptides comprising the first and/or the second bioluminescent protein of the dual-color luminescence system in cells as described in [10] item [a], [b], and [c] and the polypeptide comprising the bioluminescent protein that provides an internal reference in cells as described in [10] item [d] are expressed under control of a constitutive promoter at a similar expression level and are stable in cellula.
  • the polypeptide comprising the bioluminescent protein that provides an internal reference in cells as described in [10] item [d] is expressed at a level suitable for ratiometric measurement of luminescence by the bioluminescent protein providing information about a biological interest in the same cells.
  • the method includes detection of light, emitted in presence of the luminogenic substrate by reference and sample cells, at two or more time points.
  • the method includes detection of light emitted in presence of the luminogenic substrate by a sample cell, which has been treated with one or more chemical, biological, or physical agents.
  • this includes concomitant or successive treatment in a short period of time of all cells described in [10] item [a] to [d] and measurement of light emission from both treated and untreated cells.
  • Treatment of cells as described in [10] item [a] and [b] might be omitted, if the respective agent is known to have no significant effect on color ratios obtained with these cells.
  • Treatment of a cell as described in [10] item [c] might be omitted, if the respective agent is known to have no significant effect on emission ratios obtained with this cell.
  • the bioluminescent protein providing an internal reference in cells as described in [10] item [d] is expressed in a way that its light emission is not affected by the biological process of interest or a treatment with chemical, biological, or physical agents except for unspecific changes of protein level or enzymatic activity under the respective experimental conditions.
  • the polypeptides comprising the bioluminescent proteins in a cell as described in [10] item [c] are expressed in a way, that their emission is unaffected by the biological process of interest or a treatment with chemical, biological, or physical agents except for unspecific changes of protein level or enzymatic activity under the respective experimental conditions, ideally are comprised in the cell at a constant ratio.
  • more than one reference cell as described in [10] item [a] or [b] may be used and color ratios for calculating emission ratios for cells as described in [10] item [c] or [d] determined using mathematical procedures such as calculating an average value or establishing a calibration curve.
  • more than one reference cell as described in [10] item [c] may be used and a reference emission ratio determined using mathematical procedures such as calculating an average value or establishing a calibration curve.
  • emission ratios for cells as described in [10] item [c] and [d] may be determined using other mathematical procedures for spectral unmixing, such as an algorithm used for image analysis.
  • the inventive dual-color luminescence system is a reporter system that can be used for instance as basic quantification tool or as read-out for sensors and bioassays. It allows to measure luminescence generated in cellula relative to an internal reference, largely abolishing problems associated with measurements of absolute levels of luminescence generated in cellula and the need for determination of a reference with a different technique for comparison of light emission from different samples.
  • the inventive dual-color luminescence system comprising the bioluminescent protein with amino acid sequence SEQ ID NO: 34 (Cluc-9) and the bioluminescent fusion protein with amino acid sequence SEQ ID NO: 41 (Yluc-12) in combination with the luminogenic substrate coelenterazine provides a superior spectral separation and a very constant emission ratio over time from cells comprising both bioluminescent proteins at a constant ratio. It thus offers a wide measurement range and is particularly suitable for conducting time course experiments.
  • the inventive 'method to conduct ratiometric measurements of luminescence using a dualcolor luminescence system' provides a procedure to conduct ratiometric measurements of luminescence, which allows to minimize the influence of experimental conditions, unspecific effects resulting from treatments with chemical, biological, or physical agents, and differences in enzymatic properties of the bioluminescent proteins. It furthermore allows to conduct time course experiments.
  • the inventive BRET system is a reporter system that can be used for instance as read-out for sensors and bioassays. Compared to BRET systems containing commonly used YFP variants derived from avGFP as acceptor, it provides a wider dynamic range of measurements due to higher BRET efficiency. It is particularly suitable to detect interactions with unstable polypeptides due to fast fluorophore maturation of the acceptor.
  • the inventive BRET system with interacting components provides a much higher sensitivity compared to a BRET system without affinity between luciferase and acceptor and is thus particularly suitable as read-out for bioassays.
  • the inventive yellow fluorescent proteins can be used for instance as fluorescent reporter, including use as donor or acceptor for resonance energy transfer.
  • a yellow fluorescent protein with SEQ ID NO: 17 (YFP A ) is particularly suitable for applications where high photostability is desirable, while a yellow fluorescent protein with SEQ ID NO: 15 (YFP B ) is particularly suitable for applications where low photostability is desirable.
  • a yellow fluorescent protein with SEQ ID NO: 15 (YFP B ) is particularly useful as acceptor for BRET in combination with Renilla luciferase, due to its interaction with the luciferase.
  • a yellow fluorescent protein with SEQ ID NO: 16 (YFP B-N ) is particularly useful as acceptor for BRET in combination with Renilla luciferase, if an affinity between luciferase and acceptor is not desired, and as acceptor for BRET in combination with NIuc.
  • the inventive bioluminescent protein with amino acid sequence SEQ ID NO: 34 can be used for instance as luminescent reporter, including use as component of a DCL or a BRET system, and is particular suitable for applications, where a blue-shifted emission is desirable.
  • inventive bioluminescent fusion proteins with amino acid sequence SEQ ID NO: 41 (Yluc-12) and SEQ ID NO: 42 (Yluc-13) can be used for instance as luminescent reporter, including use as component of a DCL system, and are particular suitable for applications, where a red-shifted emission with a narrow emission peak is desirable.
  • the inventive 'method to obtain enhanced fluorescent proteins using measurements of BRET efficiency in test fusion proteins and of relative fluorescence intensity 1 can be used to develop fluorescent proteins, BRET systems, and bioluminescent fusion proteins with enhanced properties.
  • Preferred areas of use of the claimed inventions are research, development, analytics, and diagnostics in biotechnology, medicine, pharmacology, agrotechnology, and environmental protection.
  • SC-Q medium containing yeast nitrogen base w/o amino acids and ammonium sulfate (CYN4201 , Formedium), 2 % glucose, 10 mM L- glutamine, and CSM-His,Leu,Trp,Ura (DCS1389, Formedium).
  • SCR-Q medium contained 2 % D(+)-raffinose pentahydrate (83400, Fluka) instead of glucose.
  • Media for microscopy SC-Q (If) and SCR-Q (If) contained low fluorescence yeast nitrogen base w/o amino acids, ammonium sulfate, folic acid and riboflavin (CYN6201 , Formedium).
  • Coelenterazine (4094.4, Roth) was dissolved at 1 mM in (methanol + 3 mM HCI) and used at a final concentration of 2 pM, if not indicated otherwise.
  • Furimazine solution NaBRET Nano-Gio Substrate, N1571 , Promega
  • Rapamycin was dissolved at 1 mg/ml in DMSO and used at a final concentration of 1 pg/ml.
  • Constructs were expressed from a genomic locus except for those marked with (epi), which were expressed from vector pRS 416. Sequences supplied contain nucleic acid sequences encoding Rluc or NIuc for lucx, SYFP2 (aa 2-238) for YFP X , Yluc-12 for Yluc x ,
  • Constructs for measurements with a YFP variant of interest were generated by replacing the sequence encoding SEQ ID NO: 21 (amino acid 2-236 of SYFP2) in the expression cassettes SYFP2-Rluc, SYFP2-Nluc, Rluc-SYFP2, Nluc-SYFP2, SYFP2-Rluc-deg, SYFP2- Nluc-deg, SYFP2-P2A 2 -Rluc, SYFP2-P2A 2 -Nluc, or mCherry-P2A 2 -SYFP2 (SEQ ID NO: 30, 57, 58, 59, 60, 61 , 62, 63 or 65), which encode the corresponding test constructs with amino acid sequence SEQ ID NO: 3, 4, 5, 6, 7, 8, 9, 10 and 11 , respectively, with a sequence encoding amino acid 2-236 of the respective YFP variant or a sequence encoding amino acid 2-234 of mNeonGreen (Shaner et a/., (2013), Nat Methods
  • SYFP2-Rluc-deg and SYFP2-Nluc-deg contain a degron with amino acid sequence SEQ ID NO: 26 (Aviram et al., (2008), Molecular and Cellular Biology, 28(22): 6858-6869).
  • SYFP2-P2A 2 -Rluc, SYFP2-P2A2-Nluc, and mCherry-P2A2-SYFP2 contain peptide P2A 2 (SEQ ID NO: 27), which contains two copies of the 2A peptide of Porcine teschovirus-1 (Souza-Moreira et a/., (2016), FEMS Yeast Res, 18(5)).
  • test construct SYFP2-P2A S 2-Rluc was used instead of SYFP2-P2A2-Rluc, which was expressed from a episomal locus and contains peptide P2A S 2 (SEQ ID NO: 28) instead of P2A 2 .
  • Test constructs containing a 3FLAG peptide instead of SYFP2 such as 3FLAG-Rluc (SEQ ID NO: 12), 3FLAG-Nluc (SEQ ID NO: 13), and mCherry-P2A 2 -3FLAG (SEQ ID NO: 14) were expressed using expression constructs such as SEQ ID NO: 29, 56 and 66, and used as donor reference for BRET measurements and to correct for background fluorescence.
  • Luminescence was measured at 30 °C using white 96-well plates (3912, Corning) and a Mithras LB 940 microplate reader (Berthold) equipped with luminescence filters 460/60 (FF01-460/60-25, Semrock), 470/40 (470QM40, Omega Optical), 525/30 (FF01 -525/30-25, Semrock), and 535/30 (535QM30, Omega Optical).
  • the initial development of new YFP variants in combination with Rluc was conducted using filters 470/40, 525/30, and 535/30.
  • the subsequent characterization in combination with Rluc and NIuc was conducted using filters 460/60, 525/30, and 535/30.
  • Filter 535/30 served as principal filter for recording acceptor emission and filter 525/30 as additional filter for determining color values.
  • the development and application of novel bioluminescent proteins and dual-color luminescence systems was conducted using filters 460/60 and 535
  • Purified proteins were analyzed by size exclusion chromatography using a Superdex 75 10/300 GL column (17-5174-01, GE Healthcare) connected to an Akta Explorer FPLC system (GE Healthcare) and 50 mM Na-P (pH 7.5), 150 mM NaCI as running buffer at a flow rate of 0.7 ml/min. Elution of the fluorescent protein was detected via absorbance at 510 nm and the column calibrated with a gel filtration standard (151 -1901 , Bio-Rad).
  • cells expressing mCherry-P2A2-YFP or reference mCherry-P2A2-3FLAG were grown over night to high density in SC-Q (If) medium, diluted 1 :80 in SCR-Q (If), and grown for at least an additional 4 h.
  • ODeoo was adjusted to 0.22 and 120 pl culture distributed to clear-bottom 96-well plates (89621 , Ibidi) at 37 °C.
  • Cells were mixed with 80 pl SCR-Q (If) containing 1 % low melting agarose (dissolved at 90 °C and cooled to 40 °C). Agarose was allowed to solidify during centrifugation in a swing-out rotor (20 min, 1300 rpm, 18 °C).
  • Brightness and photostability of intracellular fluorescence were analyzed using an Axioplan Z1 microscope equipped with a mercury lamp (HXP 120c) and Axiovision software (Zeiss). Images for brightness measurements were taken with a 20x objective (420650-9901 , Zeiss) using filter set 43HE for RFP fluorescence (autofocus, 3 s acquisition) and 46HE for YFP fluorescence (2 s acquisition). YFP bleaching curves were recorded with a 40x objective (420460-9900, Zeiss) using filter set 46HE by manually focusing cells (VIS) and capturing images for 1 s every 10 s over 1 min under continuous illumination.
  • VIS manually focusing cells
  • Image analysis was done using Imaged software. From the first image a background region was selected and an ROI containing all cells was obtained with commands threshold (default) / create selection / add to manager. YFP/mCherry ratios were corrected for background fluorescence obtained with the mCherry reference and intracellular brightness calculated relative to average values for SYFP2 from three experiments.
  • Luminescence spectra of bioluminescent proteins were recorded using a LS 55 fluorescence spectrometer (Perkin Elmer) with cultures of cells expressing the respective proteins in SC-Q medium lacking riboflavin and containing 5
  • YFP variants derived from avGFP which exhibit enhanced BRET efficiency, have been developed via mutagenesis and determination of BRET ratios and color values using test fusion proteins as described in [1], This led unexpectedly to a strongly increased BRET efficiency that could be attributed in part to an interaction between luciferase and acceptor. Further development of the YFP thus aimed at achieving an optimal interaction between luciferase and acceptor characterized by a very high BRET efficiency in fusion proteins and a low background interaction between separately expressed proteins. In addition, the development of an enhanced YFP variant without affinity for Rluc has been pursued as well, which unexpectedly yielded a YFP variant particularly suitable as acceptor for BRET with Niue. Finally, a bright photostable YFP variant for use in fluorescence microcopy has been developed using in addition measurements of fluorescence intensity in cellula as described in [1].
  • SYFP2 Based on an initial comparison of previously disclosed YFP variants with an amino acid sequence derived from SEQ ID NO: 1 (avGFP), SYFP2 with amino acid sequence SEQ ID NO: 2 corresponding to avGFP (F46L, F64L, S65G, S72A, M153T, V163A, S175G, T203Y, A206K) has been selected as starting point for optimization.
  • avGFP amino acid sequence derived from SEQ ID NO: 1
  • the YFP portion of test fusion protein SYFP2-Rluc has been optimized by targeted mutagenesis to increase BRET efficiency while maintaining the spectral properties of the fluorescent protein.
  • variant YFP B (SEQ ID NO: 15) corresponding to avGFP (S30R, Y39I, F46V, F64V, S65G, S72A, F84Y, F99S, N105K, E142A, Y145F, M153T, V163A, S175G, T203Y, A206R, L207V, S208Y, V219A, L220V, L221 I, V224M), which yielded a BRET ratio of 39 compared to 3.4 obtained with SYFP2.
  • the increase in BRET efficiency was accompanied by a shift of the emission maximum of YFP B relative to those of SYFP2 to a shorter wavelength (Table II).
  • Table II Development of YFP B (#17) via mutagenesis of SYFP2 (#1 ). Shown are BRET ratios (BR) and color values (CV) obtained for YFP-Rluc fusion proteins and BRET ratios obtained for YFP-P2A S 2-Rluc constructs containing different YFP variants. Clz has been used at a final concentration of 5 pM.
  • YFP B XY refers to the amino acid present in position 206 (X) and 221 (Y) with YFP B RI being identical to YFP B , and YFP B AI being identical to YFP B D .
  • avGFP S30R, Y39I, F46V, F64V, S65G, S72A, F84Y, F99S, N105K, E142A, Y145F, M153T, V163A, S175G, T203Y, L207V, S208Y, V219A, L220V, L221 I,
  • mutation Q204R meant to disrupt either interaction was introduced into YFP B D to create variant YFP B N (SEQ ID NO: 16) corresponding to avGFP (S30R, Y39I, F46V, F64V, S65G, S72A, F84Y, F99S, N105K, E142A, Y145F, M153T, V163A, S175G, T203Y, Q204R, L207V, S208Y, V219A, L220V, L2211, V224M).
  • YFP B D eluted as dimer
  • variants thereof carrying mutations A206R (YFP B ) or Q204R (YFP B N ) eluted as monomers like mVenus, which contains mutation A206K commonly used for monomerization (Fig. 2).
  • Brightness and photostability of the new variants in cellula were analyzed using wide-field fluorescence microscopy with cells containing constructs encoding mCherry-P2A2-YFP, which allow for co-expression of YFP variants with the red fluorescent protein mCherry.
  • YFP B and YFP B N displayed enhanced brightness relative to mCherry compared to SYFP2, but low photostability under continuous illumination.
  • the DNA sequence encoding YFP B was thus further mutagenized to generate a variant with enhanced photostability.
  • YFP A (SEQ ID NO: 17) corresponding to avGFP (S30R, Y39I, L42F, F46L, F64V, S65G, S72A, F84Y, F99S, N105K, 1123V, E142A, Y145F, M153T, R168K, S175G, Y182V, T203Y, A206R, L207I, S208Y, V219A, L220I, L221 I, V224I), YFP A N (SEQ ID NO: 51 ) corresponding to avGFP (S30R, Y39I, L42F, F46L, F64V, S65G, S72A, F84Y, F99S, N105K, 1123V, E142A, Y145F, M153T, R168K, S175G, Y182V, T203Y, Q204R, L207I, S208Y, V219A,
  • Fusion proteins YFP-Rluc, Rluc-YFP, YFP-Nluc, and Nluc-YFP containing YFP B , YFP B-N , YFP A , or YFP A N exhibited enhanced BRET efficiency compared to fusion proteins containing SYFP2 or the other YFP variants tested, confirming that the new YFP variants are superior BRET acceptors, with YFP A and YFP A-N being somewhat less effective than YFP B and YFP B N .
  • fusion proteins containing NIuc YFP B-N and YFP A-N exhibited higher BRET efficiency compared to YFP B and YFP A , respectively (Table VI).
  • YFP B and YFP A exhibited strongly enhanced BRET efficiency compared to SYFP2 specifically in YFP-Rluc fusion proteins. This was several fold higher than the BRET efficiency obtained with fusion proteins Rluc-YFP, YFP-Nluc, and Nluc-YFP containing the same YFP variants (Table VI).
  • YFP-P2A2-Rluc In measurements with constructs encoding YFP-P2A2-Rluc a low level of association between Rluc and separately produced YFP B or YFP A could be detected, but no interaction between Rluc and YFP B N or YFP A N .
  • the energy transfer measured with YFP-P2A2-Nluc constructs may be due to random proximity of donor and acceptor or the presence of small amounts of fusion protein (Table VI).
  • Table VI Comparison of YFP variants derived from avGFP. Shown are BRET ratios, BRET efficiency relative to similar fusion proteins containing SYFP2 as acceptor (BE), and color values obtained with different test constructs containing Rluc or NIuc.
  • the fraction of mature YFP in YFP-Nluc-deg has been calculated assuming a similar quantum yield for NIuc and YFP-Nluc and complete maturation of the YFP fluorophore in fusion proteins without degron.
  • the presence of a physical interaction between the Rluc and certain YFP variants, which causes strongly enhanced BRET efficiency can thus been concluded based on the evidence that the high BRET efficiency in a YFP-Rluc fusion protein compared to SYFP2- Rluc a) is strongly reduced in similar fusion proteins containing the unrelated luciferase NIuc, b) is strongly reduced for fusion proteins containing Rluc and YFP in the reverse order and therefore presumably subject to sterical inhibition, c) depends on the presence of a few amino acids, whose side chains are exposed at the YFP surface and are located within a surface area that could function as interface for interaction with Rluc, and d) is accompanied by a weak interaction between luciferase and YFP
  • Fusion proteins containing Rluc or NIuc yielded average color values for YFP B , YFP B-N , YFP A , and YFP A N of 1.06, 1.04, 1.03, and 1.03 respectively, which indicate a shift of the emission maximum to shorter wavelength compared to conventional YFP variants like mVenus, whose average value of 1 .21 corresponds to an emission maximum at 528 nm (Shaner et a/., (2013), Nat Methods, 10(5): 407-409) (Table VI).
  • YFP B Under continuous illumination, YFP B exhibits a lower and YFP A a higher photostability compared to the other YFP variants tested (Fig. 3B).
  • the fusion proteins YFP B -Rluc and YFP B N -Nluc provide bioluminescent fusion proteins with enhanced BRET efficiency. Shortening of the linker (including the unstructured C-terminal peptide encompassing amino acids 231 to 238 of YFP B ) in fusion protein YFP B -Rluc from 20 amino acids to 3 amino acids yielded the bioluminescent fusion protein Yluc-1 (SEQ ID NO: 20) with composition YFP B 2-23o-[ASG]-Rluc, whose BRET efficiency relative to SYFP2- Rluc exceeded the BRET efficiency of the YFP B -Rluc fusion protein (Table VII).
  • Table VII Analysis of bioluminescent fusion protein Yluc-1 . Shown are color ratios, BRET ratios, and BRET efficiency relative to SYFP2-Rluc for Yluc-1 in comparison with YFP B -Rluc.
  • Rluc and Yluc-1 provide a pair of bioluminescent proteins, which emit light with a good spectral separation using the same luminogenic substrate, the possibility has been explored to conduct ratiometric measurements by co-expressing Rluc and Yluc-1 .
  • Yluc-1 has been expressed using expression cassette Ylucx and new Yluc variants that emit light with a higher color ratio (535:460) compared to Yluc-1 have been created by targeted mutagenesis of SEQ ID NO: 18 (Rluc) comprised in SEQ ID NO: 20.
  • Cluc-7 SEQ ID NO: 33
  • Cluc-8 SEQ ID NO: 101
  • Cluc-9 SEQ ID NO: 34
  • Rluc A54P, A55T, A123S, 1140V, W153F, D154E, E155D, I166L, M185V, S218T, P220L, L225I
  • Cluc-10 SEQ ID NO: 102
  • Yluc-9 (SEQ ID NO: 39) comprising Rluc-Y9 (SEQ ID NO: 35) corresponding to Rluc (G118I, A123S, C124A, A126G, 1163V, I166L, I223V, L225I, L284Y) and Yluc-11 (SEQ ID NO: 40) comprising Rluc- Y11 (SEQ ID NO: 36) corresponding to Rluc (G118I, A123S, C124A, A126G, W153F, 1163V, I166L, K189E, I223V, L225I, L284Y) (Table IX-A).
  • Yluc- 12 (SEQ ID NO: 41 ) comprising Rluc-Y12 (SEQ ID NO: 37) corresponding to Rluc (G118I, A123S, C124A, A126G, D154E, E155D, 1163V, I166L, I223V, L225I, L284Y) and Yluc-13 (SEQ ID NO: 42) comprising Rluc-Y13 (SEQ ID NO: 38) corresponding to Rluc (G118I, A123S, C124A, A126G, W153F, D154E, E155D, 1163V, I166L, K189E, I223V, L225I, L284Y), respectively (Table IX-B).
  • the Rluc-Y luciferases comprised in Yluc-9, Yluc-11 , Yluc-12, and Yluc-13 emit light with a higher color ratio (535:460) and yield lower emission ratios (Rluc:Cluc) in combination with Cluc-9 compared to Rluc (Table IX-C).
  • Table IX Characterization of Yluc variants and the luciferase contained therein
  • a Clz was used at a final concentration of 5 ,M. _ _ Variant
  • the higher color ratios (535:460) obtained for new Yluc variants compared to Yluc-1 can thus be attributed to a moderately red-shifted emission and a lower capacity to emit light in absence of a BRET acceptor of the luciferase contained therein, and a moderately enhanced BRET efficiency of the respective Yluc variants compared to Yluc-1.
  • Table X Analysis of mutations present in Clue or Yluc variants in isolation. Shown are color ratios of individually expressed luciferases and emission ratios obtained in combination with Yluc-9, measured in presence of 5 pM Clz.
  • the effect of individual mutations incorporated into Clue or Yluc has been analyzed in isolation.
  • the blue-shifted emission of Clue variants is mostly due to mutations A54P and P220L.
  • a hypsochromic effect associated with mutations in P220 has not been disclosed previously and was also detected for P220V, P220I, and P220M.
  • mutations M185V, S218T, and L225I contribute to the blue-shifted emission of Clue variants.
  • the red- shifted emission of Rluc-Y variants is mostly due to mutations G1181, 1163V, and I223V.
  • a bathochromic effect associated with mutations in G118 has not been disclosed previously and was also detected for mutations G118V and G118L.
  • Introduction of mutations A55T, A123S, I166L, M185V, K189E, and S218T into Rluc leads to increased emission ratios in combination with Yluc-12 (Table X).
  • Table XI summarizes, which mutations contribute to the spectral properties of the new Clue and Yluc variants and which mutations have been introduced to achieve a high and relatively constant emission ratio in cells co-expressing a variant of the other bioluminescent protein.
  • Example 5 Method to conduct ratiometric measurements of luminescence using a dualcolor luminescence system
  • Color and emission ratios obtained with cells expressing bioluminescent proteins can change over time to a certain degree. This is due to effects such as changes of media absorbance due to accumulation of reaction products derived from the luminogenic substrate, different enzymatic properties of the bioluminescent enzymes, inhibition of light production or alteration of spectral properties of light emitted by bioluminescent proteins due to chemical modification of the proteins in presence of the luminogenic substrate, and the cellular response to the presence of the luminogenic substrate and compounds derived thereof. Furthermore, color and emission ratios can be affected in an unspecific way by experimental conditions, for instance by altering the intracellular pH or redox state, cellular transport processes across membranes, or protein synthesis in general.
  • the method comprises:
  • a dual-color luminescence system comprising a first bioluminescent protein B1 and a second bioluminescent protein B2, and a luminogenic substrate; wherein in presence of the luminogenic substrate B1 emits light in a first spectral range, wherein little emission from B2 is detectable; and B2 emits light in a second spectral range, wherein little emission from B1 is detectable; and wherein the second spectral range is shifted to longer wavelengths compared to the first spectral range.
  • a reference cell (R1 ) comprising B1 , whose light output in both spectral ranges provides the color ratio of light emitted by B1 under the experimental conditions.
  • a reference cell (R2) comprising B2, whose light output in both spectral ranges provides the color ratio of light emitted by B2 under the experimental conditions.
  • a reference cell (R3) comprising B1 and B2, whose light output in both spectral ranges provides information about unspecific changes in the emission ratio of both bioluminescent proteins under the experimental conditions.
  • sample cells comprising B1 and B2, whose light output in both spectral ranges provides information about the biological process of interest under the experimental conditions; wherein light emission from one of the bioluminescent proteins provides information about a biological process of interest and light emission from the other bioluminescent protein provides an internal reference.
  • Cluc-7 has been expressed under control of PADH-I, PACTI, PGLCZ, and PPKCI and Yluc-9 under control of PACTI, using expression cassettes SEQ ID NO: 69, 70, 71 , 72 and 77.
  • Cells expressing Clue and Yluc separately or in combination were analyzed by measuring luminescence in presence of Clz with filters 460/60 and 535/30 and without filter, and via immunoblotting.
  • Emission ratios (Cluc:Yluc) obtained with cells expressing Clue from a genomic locus varied little between cultures, while large deviations were observed for emission ratios from cells expressing Clue from an episomal plasmid.
  • Cells expressing Clue from an episomal locus yielded a 1.61 -fold higher average emission ratio compared to cells expressing Clue from a genomic locus 3 days after transformation and a 1 .36-fold higher average emission ratio 34 days after transformation (Fig. 7).
  • 3FLAG-tagged constructs yielded emission ratios (Cluc:Yluc) comparable to those obtained with untagged Clue and these remained constant in presence of CHX.
  • N- or C-terminal tagging of Clue with 3HA led to strongly reduced steady-state emission ratios, which declined further in presence of CHX, demonstrating that fusing a 3HA-tag to a cytoplasmic protein can render it unstable (Fig. 8).
  • Insertion of tvps?3 or tADHi transcriptional terminators before the sequence encoding sTRSV restored normal expression levels, indicating efficient transcriptional termination by both sequences (Table XII).
  • Table XII DCL analysis of efficiency of transcriptional termination. Shown are absolute and relative emission ratios (Cluc:Yluc) determined in combination with Yluc-12 for cells containing Cluc-9 expression cassettes with or without sequences functioning as transcriptional terminator, followed in some constructs by sequence encoding sTRSV ribozyme.
  • an integration cassette consisting of a Clue expression cassette (SEQ ID NO: 78) and marker gene LIRA3 was inserted at the respective genomic loci in cells containing a Yluc expression cassette (SEQ ID NO: 84) at another genomic locus.
  • an integration cassette consisting of Clue expression cassette, URA3, and Yluc expression cassette was inserted into the same genomic loci in wt cells. Cells expressing Clue and Yluc separately were used as reference to determine color values, while average emission ratios obtained for the respective experiments were used as reference to calculate relative emission ratios.
  • Table XIII DCL analysis of position effects at genomic integration sites In the first experiment, an integration cassette containing a Cluc-9 expression cassette and a marker gene (URA3) was integrated at genomic loci of interest, with a Yluc-12 expression cassette being present at another locus. In the second experiment, an integration cassette containing Clue expression cassette, marker gene and Yluc expression cassette was integrated at the same genomic loci in wt cells. Shown are total emission ratios (Cluc:Yluc) and emission ratios calculated relative to the average emission ratio obtained for each measurement of the respective experiment.
  • Cluc:Yluc total emission ratios
  • fpr1 T0R1-1 cells lacking the yeast ortholog of FKBP12 and carrying a rap-insensitive allele of TOR1 containing donor construct FRB- Rluc (expression cassette SEQ ID NO: 85) and either mock acceptor construct FKBP12 (expression cassette SEQ ID NO: 86) or acceptor construct FKBP12-YFP (expression cassette SEQ ID NO: 87 or a variant thereof encoding a different YFP variant).
  • Tpk1* Tpk1 (aa 53-397, T241A) of protein kinase A (PKA) with BRET.
  • Cells analyzed contained donor construct Rluc-TPK1* (expression cassette SEQ ID NO: 88) and acceptor construct Bcy1 -SYFP2 (expression cassette SEQ ID NO: 89) or Bcy1-YFP B (expression cassette SEQ ID NO: 90), which replaced the endogenous Bcy1 , or endogenous Bcy1.
  • raffinose-containing medium SCR-Q, a medium providing low amounts of glucose via invertase cleavage of raffinose
  • SC-Q SC- Q medium containing 2 % glucose.
  • Bcy1-YFP and Rluc-Tpk1* interacted strongly in the absence of a carbon source and much less in the presence of glucose.
  • Use of YFP B as acceptor instead of SYFP2 yielded much higher BRET values, but had little effect on the difference between low and strong binding conditions (Fig. 10A).
  • RafRBD mammalian Raf
  • Ras2 T42N Ras-binding domain of mammalian Raf
  • RafRBD Ras-binding domain of mammalian Raf
  • RafRBD Ras-binding domain of mammalian Raf
  • Ras2 T42N Ras-binding domain of mammalian Raf
  • Ras2 G19V Cells containing donor construct Rluc-RAS2 (expression cassette SEQ ID NO: 91 ) or variants thereof encoding Ras2 T42N or Ras2 G19v together with acceptor construct RafRBD-YFP B (expression cassette SEQ ID NO: 93) or mock control RafRBD-3FLAG (expression cassette SEQ ID NO: 92) were analyzed in SC-Q medium.
  • Rluc-RAS2 expression cassette SEQ ID NO: 91
  • RafRBD-YFP B expression cassette SEQ ID NO: 93
  • RafRBD-3FLAG expression cassette SEQ ID NO: 92
  • Ras2 T42N yielded lower and Ras2 G19v higher BRET values compared to wildtype Ras2, confirming that a sensor consisting of Rluc-Ras2 and RafRBD-YFP B is capable to detect changes in the nucleotide loading state of Ras2 (Fig. 11 ).
  • Example 14 Development of an enhanced luciferase for use as component of a BRET system with interacting components
  • Native Rluc yields a relatively low emission ratio compared to Cluc-9 when analyzed in combination with a Yluc variant (Table II). It would therefore be desirable to engineer a variant of Rluc for use in the BRET system with interacting components, which compared to Rluc yields a higher emission ratio (Rluc:Yluc) in cells co-expressing the Rluc variant with a Yluc variant while maintaining a high BRET ratio in a YFP B -Rluc fusion protein.
  • variant Rluc-B SEQ ID NO: 46
  • Rluc A123S, D154E, E155D, I166L
  • fusion proteins YR SEQ ID NO: 47
  • YR-B SEQ ID NO: 48
  • Cells used contained expression cassettes SEQ ID NO: 95 to 100.
  • Rluc-B emitted light with a similar color ratio as Rluc, and yielded a similar BRET ratio in the YR-B fusion protein compared to YR.
  • Rluc-B yielded an approximately three times higher emission ratio (Rluc:Yluc-12) compared to Rluc, while YR-B yielded a moderately increased emission ratio (YR:Cluc-9) compared to YR (Table XIV-B).
  • Table XIV Development of a Rluc variant with enhanced brightness.
  • Rluc-B is more suitable than Rluc for use as component of the BRET system with interacting components.
  • variants with blue-shifted emission such as Cluc-9, Cluc-10, Cluc-11 (SEQ ID NO: 49) corresponding to Rluc (A54P, A55T, A123S, 1140V, W153F, D154E, E155D, I166L, S218T, P220L, L225I), or Cluc-12 (SEQ ID NO: 103) corresponding to Rluc (A54P, A55T, A123S, 1140V, W153F, D154E, E155D, W156Y, I166L, S218T, P220L, L225I) could be used.
  • the maximal peak emission wavelength of the luciferase comprised in a BRET system does not have a strong impact on BRET values obtained in combination with the same acceptor.
  • terminus ‘comprise’ means to include, encompass or contain all of the mentioned elements or components as a minimum, without excluding additional elements or components that may further be present.
  • a preferred embodiment of the terminus ‘comprising' is ‘consisting of.
  • terminus ‘consist’ means to include, encompass or contain all of the mentioned elements or components and allows for no additional elements or components to further be present.
  • singular forms of any type of elements, features or components may be interpreted to also refer to plural forms thereof.
  • the terminus ‘about’ can encompass variations of ⁇ 10%, ⁇ 5%, ⁇ 2%, ⁇ 1 %, ⁇ 0.5%, or ⁇ 0.1 % of the numerical value of the number which the term ‘about’ modifies.
  • the terminus ‘about’ encompasses variations of less than ⁇ 5% of the numerical value of the number. More preferably, the terminus ‘about’ encompasses variations of less than ⁇ 1% of the numerical value of the number.
  • the terminus ‘in cellula’ refers to processes and observations occurring in living cells. It comprises processes and observations occurring in cells in vivo (i.e. in a living organism) and processes and observations occurring in cells cultured in vitro, except human embryonic stem cells. In a preferred embodiment, 'in cellula' does not refer to cells 'in vivo' in humans and animals.
  • the terminus ‘cell’ refers to the basic structural and functional unit of all living organisms. It is the smallest independently functioning entity that possesses the essential attributes of life, including the ability to carry out metabolism, respond to stimuli, and reproduce.
  • Examples for cells include, but are not limited to, bacterial cells, yeast cells, insect cells and mammalian cells. In an exemplary embodiment, the cells are yeast cells.
  • bacterial cell refers to single-celled prokaryotic microorganisms of the domain Bacteria that are round, spiral-shaped, or rod-shaped, which typically live in soil, water, organic matter, or the bodies of plants and animals. They are able that produce their own sustenance especially from sunlight or are saprophytic or parasitic, are often motile by means of flagella, reproduce especially by binary fission, and include many important pathogens.
  • Examples for bacterial cells to be used in the present inventive methods include, but are not limited to, cells of the species Escherichia coli, Bacillus subtilis, Caulobacter crescentus, Synechocystis sp. PCC 6803, and Streptomyces coelicolor.
  • yeast cell refers to a type of fungus that is characteristically single-celled, eukaryotic, reproduces asexually by budding or binary fission, produces ascospores and is capable of fermenting carbohydrates.
  • yeast cells to be used in the present inventive methods include, but are not limited to, cells of the species Saccharomyces cerevisiae, Schizosaccharomyces pombe, Ashbya gossypii, Pichia pastoris, and Klyveromyces lactis.
  • insect cell refers to a cell derived or isolated from any member of the class Insecta, the largest class of phylum Arthropoda.
  • examples for insect cells to be used in the present inventive methods include, but are not limited to, Sf9 cells, Sf21 cells, S2 cells and High Five cells.
  • mammalian cell refers to a cell is derived or isolated from tissue of any member of the class Mammalia.
  • a mammalian cell may be a normal tissue -derived cell or a cancer cell.
  • Examples for mammalian cells to be used in the present inventive methods include, but are not limited to, CHO cells, NSO cells, SP2/0 cells, YB2/0 cells, BHK cells, HEK293 cells, HT-1080 cells, PER.C6 cells, and Huh-7 cells.
  • 'mammalian cell' does not refer to human embryonic stem cells.
  • the terminus 'reporter' refers to a protein that can be produced in vivo and detected using conventional biological, physical or chemical techniques based on its biological activity.
  • the terminus 'reporter system' refers to a system comprising two or more reporters that are used in combination to detect or quantify a biological process of interest.
  • the terminus 'chromophore' refers to a chemical moiety that absorbs light of specific wavelengths in the visible range of the spectrum.
  • the terminus 'fluorophore' refers to a chromophore that absorbs light of specific wavelengths in the visible range of the spectrum and re-emits light at longer wavelengths in the visible range of the spectrum.
  • the terminus 'chemiphore' refers to a chemical moiety in an excited state produced via a chemical reaction that emits light in the visible range of the spectrum.
  • termini 'resonance energy transfer' or 'RET' refer to a non-radiative transfer of energy from a donor chromophore or chemiphore to an acceptor chromophore.
  • the terminus 'luminogenic substrate' refers to a chemical compound that can be used in combination with a luciferase to produce light.
  • the terminus 'luminogenic substrate comprising an imidazopyrazinone moiety' refers to a luminogenic substrate comprising an imidazo[1 ,2-a]pyrazin-3(7H)-one moiety such as 8- benzyl-6-(4-hydroxyphenyl)-2-[(4-hydroxyphenyl)methyl]imidazo[1 ,2-a]pyrazin-3(7H)-one (coelenterazine), 8-benzyl-2-(furan-2-ylmethyl)-6-phenylimidazo[1 ,2-a]pyrazin-3(7H)-one (furimazine), or 8-benzyl-2-(furan-2-ylmethyl)-6-(4-hydroxyphenyl)imidazo[1 ,2-a]pyrazin- 3(7H)-one.
  • the terminus 'bioluminescent protein' refers to a protein capable to produce light in presence of a luminogenic substrate. This can be a protein comprising a luciferase or a protein comprising a luciferase and at least one acceptor for BRET such as a bioluminescent fusion protein.
  • the terminus 'luciferase' refers to a protein, which is capable to produces light in presence of a luminogenic substrate, and which does not contain an acceptor for BRET.
  • luciferases catalyze one or more steps in the conversion of a luminogenic substrate into a chemiphore and provide a proteinaceous environment necessary for efficient light emission by the chemiphore.
  • the terminus 'donor luciferase' refers to a luciferase, which in combination with a luminogenic substrate provides a chemiphore that serves as donor for BRET, e.g. as component of a bioluminescent fusion protein or BRET system.
  • the terminus 'bioluminescent fusion protein' refers to a protein comprising a (donor) luciferase and an acceptor for BRET, wherein the luciferase is connected to the acceptor in a way allowing for efficient non-radiative energy transfer in presence of a luminogenic substrate.
  • a common parameter for assessing the quality of a bioluminescent fusion protein is the BRET ratio.
  • the terminus 'ratiometric measurement of luminescence' refers to a measurement of luminescence within two different spectral ranges, wherein luminescence is quantified based on the ratio of emission in both spectral ranges.
  • the terminus 'internal reference' refers to a signal that is provided by a composition of matter present in samples to alleviate quantitative analysis.
  • the signal is similar to a signal provided by a matter of interest but sufficiently different so that the two signals are readily distinguishable with a suitable measurement device.
  • the matter providing the internal reference is similar, but not identical to the matter of interest in the samples, as in the ideal case experimental conditions should, relative to the amount of each species, effect the internal reference in the same way as the signal from the matter of interest.
  • the signal provided by the matter of interest can be quantified relative to the internal reference, allowing in an ideal case, to correct for systematic errors, fortuitous changes in experimental conditions between measurements, and unspecific effects of experimental conditions on the intensity of the signals.
  • the matter providing the internal reference should be present at a similar amount as the matter of interest.
  • the termini 'dual-color luminescence system' or 'DCL system' refer to a reporter system comprising two bioluminescent proteins, wherein the two bioluminescent proteins produce spectrally distinct light with the same luminogenic substrate. Since not every luminogenic substrate capable to produce light with the bioluminescent proteins of a DCL system is suitable for use with the respective DCL system, the luminogenic substrate can also be considered as part of the DCL system.
  • the terminus 'BRET system' refers to a reporter system comprising a (donor) luciferase and an acceptor, wherein energy can be transferred non-radiatively from a chemiphore derived from a luminogenic substrate and contained in the luciferase to an acceptor fluorophore if luciferase and acceptor are in a suitable distance and orientation, whereupon the acceptor emits light that is spectrally distinct from light emitted by the chemiphore. Since not every luminogenic substrate capable to produce light with the luciferase is suitable for use with the respective BRET system, the luminogenic substrate can also be considered as part of the BRET system.
  • terminus 'BRET system with interacting components' refers to a BRET system, wherein (donor) luciferase and acceptor interact in a way that places the chemiphore contained in the luciferase and the fluorophore contained in the acceptor in a distance and orientation allowing for efficient energy transfer.
  • a high BRET efficiency observed for a fusion protein comprising both donor luciferase and acceptor of the respective BRET system a) is specific for the particular combination of luciferase and acceptor, meaning that BRET efficiency is strongly reduced in similar fusion proteins containing an unrelated luciferase and/or an unrelated acceptor, b) depends on the presence of a few amino acids, whose side chains are exposed at the surface of the donor and/or acceptor and are located within a surface area that could function as interface for interaction with the other protein, and c) is accompanied by an interaction between donor luciferase and acceptor that is detectable if both proteins are produced separately at a relatively high level.
  • the terminus 'Renilla luciferase' refers to a luciferase from an organism of the genus Renilla, such as Renilla reniformis or Renilla mulleri, including engineered variants derived thereof.
  • the terminus 'Rluc' refers to the luciferase from Renilla reniformis with amino acid sequence SEQ ID NO: 18.
  • terminus 'Niue' refers to a luciferase with amino acid sequence SEQ ID NO: 19.
  • the terminus 'avGFP' refers to the green fluorescent protein from Aequoria victoria with amino acid sequence SEQ ID NO: 1.
  • the terminus 'fluorescent protein' refers to a protein with the characteristic p-barrel structure of avGFP, wherein amino acids within the central a-helix autocatalytically form a fluorophore in a process called maturation.
  • Mature fluorescent proteins absorb light of specific wavelengths in the visible range of the spectrum and re-emit light at longer wavelengths in the visible range of the spectrum.
  • Fluorescent proteins are commonly distinguished from the structurally similar 'chromoproteins', wherein amino acids within the central a-helix autocatalytically form a chromophore that absorbs light, but does not emit significant amounts of light.
  • the terminus ‘derived from’ in the context of amino acid sequences means that a first amino acid sequence is based on a second (reference) amino acid sequence, i.e. the first amino acid sequence has been created from the second amino acid sequence by e.g. amino acid mutations, additions, and/or deletions, and that the first amino acid sequence has at least 60 % identity to the second amino acid sequence.
  • the denotation of mutations in the first amino acid sequence indicates changes of amino acids at the respective positions of the second amino acid sequence.
  • termini 'yellow fluorescent protein', 'yellow fluorescent protein derived from Aequoria victoria GFP', 'YFP', or 'YFP derived from avGFP' refer to a fluorescent protein with red-shifted emission compared to avGFP, whose amino acid sequence has been derived from SEQ ID NO: 1 and contains mutation T203Y.
  • the terminus YFP B 2-23o' refers to a yellow fluorescent protein with amino acid sequence SEQ ID NO: 23.
  • the termini 'color ratio', 'color ratio (X:Y)', 'CR', and 'CR (X:Y)' refer to the ratio between the amount of light detected in a first spectral range X and the amount of light detected in a second spectral range Y, wherein the wavelengths within the first spectral range are longer than the wavelengths within the second spectral range.
  • Data in brackets indicate the first (X) and the second (Y) spectral range, with numerical data referring to the median wavelength of the respective spectral range.
  • the terminus 'spectral separation' refers to the ratio between the color ratios of light emitted by a first and a second bioluminescent protein in presence of the same luminogenic substrate, wherein the first bioluminescent protein emits light with a maximum emission peak at longer wavelengths compared to the second.
  • termini 'emission ratio', 'emission ratio (A:B)', 'ER', and 'ER (A:B)' refer to the ratio between the amount of light emitted by bioluminescent protein A and detected in a spectral range suitable to detect emission from bioluminescent protein A, wherein little emission from bioluminescent protein B is detectable, and the amount of light emitted by bioluminescent protein B and detected in a different spectral range suitable to detect emission from bioluminescent protein B, wherein little emission from bioluminescent protein A is detectable, determined for cells co-expressing both bioluminescent proteins.
  • termini 'BRET ratio', 'BRET ratio (X:Y)', 'BR', and 'BR (X:Y)' refer to the ratio between a color ratio (X:Y) obtained for a sample cell comprising a luciferase and an acceptor for BRET (CRsampie) and a color ratio (X:Y) obtained for a reference cell comprising the same luciferase without acceptor (CR re f).
  • Light emission is measured in a spectral range X suitable to detect emission from the acceptor (acceptor range) and a spectral range Y suitable to detect emission from the luciferase, wherein no significant emission from the acceptor is detectable (donor range). Numbers in brackets indicate the median wavelength of acceptor range and donor range.
  • the BRET value obtained for a cell comprising luciferase and acceptor represents the ratio between the amount of light emitted by the acceptor and detected in the acceptor range and the amount of light emitted by the luciferase and detected in the acceptor range. Numbers in brackets indicate the median wavelength of acceptor range (X) and donor range (Y).
  • the terminus 'expression cassette' refers to a nucleic acid, which comprises a promoter, a nucleic acid encoding a polypeptide, and a transcription terminator, wherein promoter and terminator are operationally linked to the coding sequence to allow for expression of the polypeptide in a cell.
  • Expression cassettes may comprise additional elements such as a nucleic acid sequence, whose presence affects the expression of the encoded polypeptide, and/or lack an element such as a terminator for functional analysis of the respective element.
  • the terminus 'test construct' refers to a polypeptide used to conduct comparative measurements with different variants of a protein of interest in cellula. It comprises the protein of interest, a reference variant thereof, or a mock control.
  • test fusion protein' refers to a test construct used to conduct measurements of BRET efficiency with different acceptors in combination with a luciferase.
  • Test fusion proteins contain a luciferase, an acceptor for BRET, and an invariant linker that separates luciferase and acceptor, and may contain additional invariant polypeptides at their N- and/or C-terminus comprising for instance a degron.
  • the termini 'test fusion protein based on SEQ ID NO: 3' or 'test fusion protein similar to SEQ ID NO: 3' refer to a polypeptide with amino acid sequence SEQ ID NO: 3 (SYFP2-Rluc); or a polypeptide derived thereof, wherein amino acid sequence SEQ ID NO: 22 has been replaced by the corresponding amino acid sequence (based on an alignment with SEQ ID NO: 22) of another YFP variant derived from avGFP (YFP-Rluc).
  • the terminus 'test fusion protein based on SEQ ID NO: 3 containing (the) luciferase' luc refers to a polypeptide derived from amino acid sequence SEQ ID NO: 3, wherein amino acid sequence SEQ ID NO: 18 has been replaced by the amino acid sequence of luc (SYFP2-luc); or a polypeptide derived thereof, wherein in addition amino acid sequence SEQ ID NO: 22 has been replaced by the corresponding amino acid sequence (based on an alignment with SEQ ID NO: 22) of another YFP variant derived from avGFP (YFP-luc).
  • the termini 'peptide' and 'polypeptide' are used interchangeably for chains of amino acids of any length.
  • 'Peptide' refers preferentially to short unstructured chains of amino acids and 'polypeptide' refers preferentially to longer chains of amino acids that adopt at least in part an ordered three-dimensional structure.
  • the terminus 'linker 1 refers to a peptide or polypeptide used to connect polypeptides in a fusion protein.
  • Linker sequences are typically 2 to 30 amino acids in length.
  • terminus 'degron' refers to a peptide or polypeptide, which reduces the stability of polypeptides comprising them in cellula.
  • the terminus 'targeting sequence' refers to a peptide or polypeptide, which directs localization of polypeptides comprising them to a cellular compartment.
  • Targeting sequences include, but are not limited to, a nuclear localization sequence, a nucleolar localization signal sequence, a nuclear export sequence, an endoplasmic reticulum localization sequence, a peroxisome localization sequence, or a mitochondrial localization sequence.
  • the terminus '% identity of a first (query) amino acid sequence to a second (subject) amino acid sequence means the value determined by comparing two optimally aligned amino acid sequences over a comparison window, wherein the portion of the query amino acid sequence in the comparison window may comprise additions or deletions as compared to the subject sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences.
  • the percentage is calculated by determining the number of positions at which the identical amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the result by 100 to yield the percentage of sequence identity.
  • % identity of an amino acid sequence to a reference sequence refers to the entire length of the reference sequence and implies that the basic functionality of the protein with the reference sequence (e.g. fluorescence for a fluorescent protein or bioluminescence for a bioluminescent protein) is maintained by the protein with the respective amino acid sequence, while aspects thereof may be changed/improved (e.g. intensity, emission wavelength, excitation wavelength etc.).
  • a method comprising the determination of a BRET ratio and a color value via measurement of light emission in three different spectral ranges in cellulo from a luciferase and a test fusion protein comprising the same luciferase and a fluorescent protein.
  • a yellow fluorescent protein containing mutation T203Y with an amino acid sequence derived from SEQ ID NO: 1 which
  • (i) comprises at least two of the following mutations: L42F, T43K, F46V, F64V, F84Y, 1123V, E142A, R168K, Y182V, Q204V, Q204R, A206R, L207V, L207I, S208Y, V219A, L220V, L220I, L221 I, V224M, and V224I; and
  • (ii) comprises at least one of the following mutations: S30R, Y39I, F46L, S65G, S72A, F99S, N105K, Y145F, M153T, V163A, and S175G.
  • the yellow fluorescent protein of item 2 wherein the amino acid sequence of the yellow fluorescent protein comprises an amino acid sequence with at least 80 % identity to any one of the amino acid sequences SEQ ID NOs: 23, 24, or 25.
  • the yellow fluorescent protein of item 2 wherein the amino acid sequence of the yellow fluorescent protein comprises SEQ ID NOs: 23, 24, or 25.
  • a BRET system wherein the acceptor comprises a yellow fluorescent protein of any one of item 2 to 5.
  • a bioluminescent fusion protein which comprises a yellow fluorescent protein of any one of item 2 to 5.
  • bioluminescent fusion protein of item 9 wherein the donor luciferase produces light with a luminogenic substrate that comprises an imidazo[1 ,2-a]pyrazin-3(7H)-one moiety.
  • bioluminescent fusion protein of item 9 wherein the donor luciferase comprises an amino acid sequence with at least 80 % identity to SEQ ID NOs: 18 or 19.
  • a cell comprising the yellow fluorescent protein of any one of items 2 to 5, the donor luciferase and the acceptor of a BRET system of any one of items 6 to 8, the bioluminescent fusion protein of any one of items 9 to 11 , or the nucleic acid or combination of nucleic acids of item 12.
  • (i) comprises at least two of the following mutations: A54P, 1140V, W153F, W156F, S218T, P220V, P220I, P220L, P220M, and L225I; and at least one of the following mutations: A55T, A123S, D154E, E155D, I166L, and M185V; wherein
  • the bioluminescent protein emits less light in cellulo in presence of coelenterazine in the spectral range between 520 and 550 nm relative to emission in the spectral range between 430 and 490 nm compared to a protein with amino acid sequence SEQ ID NO: 18.
  • bioluminescent protein of item 1 wherein the amino acid sequence of the bioluminescent protein has at least 80 % identity to SEQ ID NO: 34.
  • bioluminescent protein of item 1 wherein the bioluminescent protein has amino acid sequence SEQ ID NO: 34.
  • (i) comprises a yellow fluorescent protein comprising an amino acid sequence with at least 80 % identity to SEQ ID NO: 23;
  • (ii) comprises a luciferase with an amino acid sequence derived from SEQ ID NO: 18 comprising at least two of the following mutations: G118V, G118I, G118L, A126G, 1163V, K189E, I223V, and L225I; and at least one of the following mutations: A123S, C124A, W153F, D154E, E155D, I166L, and L284Y; wherein
  • the bioluminescent fusion protein emits more light in cellulo in presence of coelenterazine in the spectral range between 520 and 550 nm relative to emission in the spectral range between 430 and 490 nm compared to a protein with amino acid sequence SEQ ID NO: 20.
  • bioluminescent fusion protein of item 4 wherein the amino acid sequence of the luciferase has at least 80 % identity to any one of SEQ ID NOs: 37 or 38.
  • bioluminescent fusion protein of item 4 wherein the luciferase has any of the amino acid sequences SEQ ID NOs: 37 or 38.
  • a dual-color luminescence system comprising
  • a first bioluminescent protein which comprises a luciferase that can be expressed intracellularly and whose activity is not regulated by binding of calcium ions to calcium-binding motifs in the luciferase;
  • a second bioluminescent protein which is a bioluminescent fusion protein comprising a luciferase with an amino acid sequence that has at least 60 % identity to the luciferase comprised in the first bioluminescent protein and at least one fluorescent protein;
  • the first and the second bioluminescent protein produce light with the same luminogenic substrate comprising an imidazopyrazinone moiety; and wherein (iv) the second bioluminescent fusion protein emits in cellulo in presence of the luminogenic substrate at least 30-fold more light in the spectral range between 520 and 550 nm relative to emission in the spectral range between 430 and 490 nm compared to the first bioluminescent fusion protein.
  • the first bioluminescent protein comprises an amino acid sequence with at least 80 % identity to SEQ ID NO: 18 or a bioluminescent protein of any one of items 1 to 3;
  • the second bioluminescent protein comprises an amino acid sequence with at least 80 % identity to SEQ ID NO: 20 or a bioluminescent fusion protein of any one of items 4 to 6.
  • a cell comprising the protein of any one of items 1 to 6, the first and the second bioluminescent protein of the dual-color luminescence system of any one of items 7 or 8, or the nucleic acid or combination of nucleic acids of item 9.
  • a method comprising the determination of an emission ratio via measurement of light emission in presence of a luminogenic substrate in two different spectral ranges from a cell comprising the first bioluminescent protein, a cell comprising the second bioluminescent protein, and a cell comprising both the first and the second bioluminescent protein of a dual-color luminescence system of any one of items 7 or 8.
  • a method of item 11 which comprises
  • a reference cell comprising both bioluminescent proteins in a way that their emission ratio provides a reference for emission ratios obtained with the sample cell(s), and (iv) one or more sample cells comprising one of the bioluminescent proteins in a way that its emission yields information about a biological process of interest and the other bioluminescent protein in a way that it provides an internal reference or that its emission yields information about the same ora different biological process of interest;
  • a yellow fluorescent protein with an amino acid sequence derived from SEQ ID NO: 1 containing mutation T203Y which comprises in addition at least two of the following mutations: L42F, T43K, F46V, F64V, F84Y, 1123V, E142A, R168K, Y182V, Q204V, Q204R, A206R, L207V, L207I, S208Y, V219A, L220V, L220I, L221 I, V224M, and V224I.
  • the yellow fluorescent protein of item 1 further comprising at least one of the following mutations: S30R, Y39I, F46L, S65G, S72A, F99S, N105K, Y145F, M153T, V163A, and S175G.
  • the yellow fluorescent protein of item 1 or 2 wherein the amino acid sequence of the yellow fluorescent protein comprises an amino acid sequence with at least 80 % identity to any one of SEQ ID NOs: 23 or 25.
  • the luciferase of item 6 further comprising at least one of the following mutations: A55T, A123S, D154E, E155D, I166L, and M185V.
  • bioluminescent protein emits less light in cellula in presence of coelenterazine in the spectral range between 520 and 550 nm relative to emission in the spectral range between 430 and 490 nm compared to a protein with amino acid sequence SEQ ID NO: 18.
  • luciferase of any of the items 6 to 9, wherein the luciferase has amino acid sequence SEQ ID NO: 34, 49, 102 or 103.
  • a bioluminescent fusion protein which comprises the yellow fluorescent protein of any of the items 1 to 5 and a luciferase.
  • bioluminescent fusion protein of item 11 wherein the luciferase produces light with a luminogenic substrate that comprises an imidazo[1 ,2-a]pyrazin-3(7H)-one moiety.
  • bioluminescent fusion protein of item 14 wherein the amino acid sequence of the luciferase further comprises at least one of the following mutations: A123S, C124A, W153F, D154E, E155D, I166L, and L284Y. 16.
  • bioluminescent fusion protein of any of the items 14 to 17, wherein the luciferase has amino acid sequence SEQ ID NO: 37 or 38.
  • a dual-color luminescence system comprising two bioluminescent proteins that produce spectrally distinct light with the same luminogenic substrate, wherein
  • the first bioluminescent protein comprises a luciferase that can be expressed intracellularly and whose activity is not regulated by binding of calcium ions to calcium-binding motifs in the luciferase;
  • the second bioluminescent protein comprises a bioluminescent fusion protein comprising a luciferase and at least one fluorescent protein
  • the amino acid sequence of the luciferase comprised in the second bioluminescent protein has at least 60 % identity, preferably at least 80 % identity to the amino acid sequence of the luciferase comprised in the first bioluminescent protein;
  • the luminogenic substrate comprises an imidazo[1,2-a]pyrazin-3(7H)-one moiety.
  • a BRET system comprising a luciferase and an acceptor for BRET, wherein the acceptor comprises the yellow fluorescent protein of any of the items 1 to 5.
  • a cell comprising the yellow fluorescent protein of any of the items 1 to 5, the luciferase of any of the items 6 to 10, the bioluminescent fusion protein of any of the items 11 to 18, the first and the second bioluminescent protein of the dual-color luminescence system of any of the items 19 to 23, the luciferase and the acceptor of the BRET system of any of the items 24 to 27, or the nucleic acid or combination of nucleic acids of item 28.
  • a method comprising the determination of an emission ratio via measurement of light emission in presence of the same luminogenic substrate in two different spectral ranges from a cell comprising the first bioluminescent protein, a cell comprising the second bioluminescent protein, and a cell comprising both the first and the second bioluminescent protein of a dual-color luminescence system of any of the items 19 to 23.
  • sample cells comprising one of the bioluminescent proteins in a way that its emission yields information about a biological process of interest and the other bioluminescent protein in a way that it provides an internal reference or that its emission yields information about the same or a different biological process of interest;
  • a yellow fluorescent protein containing mutation T203Y with an amino acid sequence derived from SEQ ID NO: 1 which
  • (i) comprises at least two of the following mutations: L42F, T43K, F46V, F64V, F84Y, 1123V, E142A, R168K, Y182V, Q204V, Q204R, A206R, L207V, L207I, S208Y, V219A, L220V, L220I, L221 I, V224M, and V224I; and
  • (ii) comprises at least one of the following mutations: S30R, Y39I, F46L, S65G, S72A, F99S, N105K, Y145F, M153T, V163A, and S175G.
  • the yellow fluorescent protein of item 1 wherein the amino acid sequence of the yellow fluorescent protein comprises an amino acid sequence with at least 80 % identity to any one of SEQ ID NOs: 23, 24 or 25.
  • the yellow fluorescent protein of item 1 wherein the yellow fluorescent protein displays enhanced BRET efficiency compared to SYFP2 in a test fusion protein based on SEQ ID NO: 3.
  • the yellow fluorescent protein of item 1 wherein the amino acid sequence of the yellow fluorescent protein comprises SEQ ID NO: 23, 24 or 25.
  • (i) comprises at least two of the following mutations: A54P, 1140V, W153F, W156F, S218T, P220V, P220I, P220L, P220M, and L225I; and at least one of the following mutations: A55T, A123S, D154E, E155D, I166L, and M185V; wherein
  • the luciferase emits less light in cellula in presence of coelenterazine in the spectral range between 520 and 550 nm relative to emission in the spectral range between 430 and 490 nm compared to a protein with amino acid sequence SEQ ID NO: 18.
  • a bioluminescent fusion protein which comprises the yellow fluorescent protein of any of the items 1 to 4.
  • bioluminescent fusion protein of item 8 wherein the donor luciferase produces light with a luminogenic substrate that comprises an imidazo[1 ,2-a]pyrazin-3(7H)-one moiety.
  • the donor luciferase comprises an amino acid sequence with at least 80 % identity to any one of SEQ ID NOs: 18 or 19.
  • (i) comprises a yellow fluorescent protein comprising an amino acid sequence with at least 80 % identity to SEQ ID NO: 23;
  • (ii) comprises a donor luciferase with an amino acid sequence derived from SEQ ID NO: 18 comprising at least two of the following mutations: G118V, G118I, G118L, A126G, 1163V, K189E, I223V, and L225I; and at least one of the following mutations: A123S, C124A, W153F, D154E, E155D, I166L, and L284Y; wherein
  • the bioluminescent fusion protein emits more light in cellula in presence of coelenterazine in the spectral range between 520 and 550 nm relative to emission in the spectral range between 430 and 490 nm compared to a protein with amino acid sequence SEQ ID NO: 20.
  • bioluminescent fusion protein of item 11 wherein the amino acid sequence of the donor luciferase has at least 80 % identity to SEQ ID NO: 37.
  • bioluminescent fusion protein of item 11 wherein the donor luciferase has amino acid sequence SEQ ID NO: 37 or 38.
  • a dual-color luminescence system comprising
  • a first bioluminescent protein which comprises a luciferase that can be expressed intracellularly and whose activity is not regulated by binding of calcium ions to calcium-binding motifs in the luciferase;
  • a second bioluminescent protein which is a bioluminescent fusion protein comprising a luciferase with an amino acid sequence that has at least 60 %, preferably at least 80% identity to the luciferase comprised in the first bioluminescent protein and at least one fluorescent protein;
  • the first and the second bioluminescent protein produce light with the same luminogenic substrate comprising an imidazo[1 ,2-a]pyrazin-3(7H)-one moiety.
  • the second bioluminescent protein comprises the yellow fluorescent protein of any of the items 1 to 4.
  • the first bioluminescent protein comprises the luciferase of any of the items 5 to 7;
  • the second bioluminescent protein comprises the bioluminescent fusion protein of any of the items 11 to 13.
  • a BRET system wherein the acceptor comprises the yellow fluorescent protein of any of the items 1 to 4.
  • a cell comprising the yellow fluorescent protein of any of the items 1 to 4, the luciferase of any of the items 5 to 7, the bioluminescent fusion protein of any of the items 8 to 13, the first and the second bioluminescent protein of the dual-color luminescence system of any of the items 14 to 18, the donor luciferase and the acceptor of the BRET system of any of the items 19 to 22, or the nucleic acid or combination of nucleic acids of item 24.
  • a method comprising the determination of an emission ratio via measurement of light emission in presence of a luminogenic substrate in two different spectral ranges from a cell comprising the first bioluminescent protein, a cell comprising the second bioluminescent protein, and a cell comprising both the first and the second bioluminescent protein of a dual-color luminescence system of any of the items 14 to 18.
  • the method of item 25, which comprises
  • sample cells comprising one of the bioluminescent proteins in a way that its emission yields information about a biological process of interest and the other bioluminescent protein in a way that it provides an internal reference or that its emission yields information about the same or a different biological process of interest;
  • Table XVI Exemplary combinations of mutations present in the inventive bioluminescent protein described in [3].
  • Table XVII Exemplary combinations of mutations present in the luciferase comprised in the inventive bioluminescent fusion protein described in [5].

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Abstract

L'invention concerne des procédés permettant d'obtenir des variants améliorés de protéines fluorescentes et bioluminescentes sur la base d'une mesure ratiométrique de la luminescence. L'invention concerne en outre des variants modifiés d'une protéine fluorescente jaune (YFP) dérivée de Aequoria victoria GFP, des variants de Renilla luciférase (Rluc) ayant une émission décalée vers le bleu (Clue), et des protéines de fusion bioluminescentes comprenant une protéine fluorescente jaune selon l'invention, comprenant des variants comprenant du Rluc ou un variant de Rluc ayant une émission décalée vers le rouge (Yluc). En outre, l'invention concerne des systèmes rapporteurs pour la mesure ratiométrique de la luminescence comprenant un nouveau système de luminescence double couleur (DCL) et un nouveau système BRET avec des composants interagissant. En outre, l'invention concerne un procédé pour réaliser des mesures ratiométriques de luminescence à l'aide d'un système de luminescence double couleur.
PCT/EP2023/080279 2022-10-28 2023-10-30 Nouvelles protéines fluorescentes et bioluminescentes et systèmes rapporteurs pour la mesure ratiométrique de la luminescence WO2024089297A1 (fr)

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