Circularly permuted haloalkane transferase fusion molecules

Abstract

The present invention relates to a modular polypeptide comprising a first partial effector sequence comprising a first portion of a circularly permuted HaloTag protein connected to a sensor module sequence, the sensor module being connected to a second portion of the circularly permuted HaloTag protein. The sensor module is a single polypeptide or pair of polypeptides that can undergo a conformational change from a first conformation to a second conformation in response to the presence or concentration of an analyte compound. The modular polypeptide is catalytically active in response to an environmental stimulus or in response to the interaction of the sensor pair. The present invention further relates to nucleic acid sequences encoding the modular polypeptides, and kits comprising same.

Description

[Technical field]

[0001] The present invention relates to polypeptide sequences that contain haloalkane transferase enzyme activity that is capable of regulating this enzyme activity in response to environmental stimuli. [Background technology]

[0002] Methods to integrate biochemical processes over time are of great scientific interest. These biochemical processes encompass, for example, protein-protein interactions, changes in metabolite concentrations, or subcellular (re)localization of proteins. Currently, these biochemical processes are mainly studied using real-time fluorescence microscopy with tailor-made biosensors. However, these approaches have several drawbacks. Especially in neurobiology, the experimental setup required for in vivo fluorescence imaging restricts the movement of the animal, affecting the behavior of the studied subject. Biochemical processes occurring in organs such as the intestine or the heart are complicated to resolve at the cellular level due to their inherent physiological motility. More generally, fluorescence microscopy is restricted to a limited field of view and tissue depth. Despite significant improvements in instrumentation, image processing, and biosensor design, it is still not possible to study fundamental events in intact rodent organs in vivo with a fluorescence microscope.

[0003] An alternative approach consists in the recording of biochemical processes that can be read at a later time. This recording process is defined as "integration". It results in the accumulation of irreversible marks over time, depending on the biochemical process under investigation.

[0004] "Post-hoc" evaluation of experiments is not only useful for in vivo studies, but also beneficial for cell-based assays. Especially for biochemical processes that occur infrequently, signal integration over time can improve readout by increasing the signal-to-noise ratio. In high-throughput screening, signal integration provides a snapshot of the phenomenon studied, thereby replacing long-term recordings via real-time microscopy, facilitating multiplexing and reducing costs. Finally, the use of fluorescent substrates allows for the identification and ultimately sorting of cell populations based on their metabolic / signal profiles for downstream analysis or therapy. Summary of the Invention [Problem to be solved by the invention]

[0005] Based on the above-mentioned state of the art, the object of the present invention is to provide means and methods for integrating biochemical processes over time. This object is achieved by the subject matter of the independent claims herein. [Means for solving the problem]

[0006] Based on the self-labeling protein HaloTag7, we provide a family of chemogenetic integrators, proteins engineered to react irreversibly with chloroalkane substrates in response to a given biochemical process. These integrators consist of split circular permutants of HaloTag7 that change conformation in response to a biochemical process, triggering labeling activity. In a typical experiment, the recording window is defined by the temporal presence of a chloroalkane substrate that can (covalently) label the chemogenetic integrator. In pulse-chase experiments, different labeled substrates can be used sequentially, offering the possibility of studying different sequential phenomena in the same biological sample. [Brief description of the drawings]

[0007] [Figure 1] ITC assay for measuring affinity between cpHaloΔ9mer and 9mer peptide: Representative measurements when 9mer peptide (30 mM) was titrated into cpHaloΔ9mer (0.6 mM) solution. Top: Raw ITC data after baseline subtraction of two concatenated measurements. Bottom: ΔH plotted against the molar ratio of 9mer to cpHaloΔ9mer (dots) and fitted to a one-site binding model (line). Heats of dilution of the peptides were subtracted before analysis. Kd was calculated from three independent measurements and errors represent standard deviation. [Diagram 2] Figure 1. Affinity of fluorescent HaloTag ligands with HaloTag7D106A. Catalytically dead mutant HaloTag7D106A was titrated against five different fluorescent HaloTag ligands at 25 nM in an FP binding assay (Halo-Carbopyronine, Halo-TMR, Halo-TMR-Azetidine, Halo-Oregon Green, Halo Alexa Fluor 488). The structures of the fluorescent ligands are shown in each graph. The data were fit to a one-site binding model and the derived Kd values ​​are tabulated. The data points at 0.1 nM and 1 nM correspond to values ​​without any added protein, but are placed at lower concentrations and are displayed on a logarithmic scale. All experiments were performed in triplicate and the error bars and annotated uncertainties represent standard deviations. [Diagram 3]Figure 1: Affinity of fluorogenic HaloTag ligands for HaloTag7D106A: Catalytically non-active mutant HaloTag7D106A was titrated against 25 nM of three different fluorogenic HaloTag ligands (Halo-silicon rhodamine, Halo-Janelia Fluor646 and Halo-Janelia Fluor 635) in a fluorescence intensity-based binding assay. The structures of the fluorogenic ligands are shown in each graph. The data were fit to a one-site binding model and the derived Kd values ​​are shown in the graphs. Due to the low affinity of the fluorogenic substrates, the 109 nM data point was extrapolated based on fully labeled HaloTag7 (similar to perfect binding) to improve the fit. The 1 nM data point corresponds to the value without any added protein, but is placed at a lower concentration and is displayed on a logarithmic scale. All experiments were performed in triplicate and the error bars and annotated uncertainties represent standard deviations. [Figure 4] Figure 1. Comparison of fluorescent substrate binding of HaloTag7D106A and cpHaloΔ9merD106A: The catalytically inactive mutant HaloTag7Δ9merD106A (blue) was titrated against three different fluorescent HaloTag ligands at 25 nM in an FP binding assay (Halo-TMR, Halo-TMR-azetidine, Halo-carbopyronine). The structure of each fluorescent ligand is shown on each graph. The data were fit to a one-site binding model and the derived Kd values ​​are shown on the graphs. The data points at 0.01 nM and 0.1 nM correspond to values ​​without any added protein, but are placed at lower concentrations and displayed on a logarithmic scale. The data were compared to HaloTag7D106A binding of the same fluorophore (red). Differences in binding affinity are indicated by the relative fold change in Kd values. All experiments were performed in triplicate and error bars represent standard deviation. [Diagram 5]Background labeling of cpHaloΔ9mer: Background labeling (at 37°C) of cpHaloΔ9mer (1 μM) in the presence of 2 μM fluorescent ligand was determined by gel fluorimetry. Samples were denatured and analyzed by SDS-PAGE followed by gel fluorescence scanning (top; gels are false-colored to highlight faint bands). Intensity was normalized to HaloTag7 labeled with the respective fluorophore (bottom). [Figure 6] FIG. 14. Optimization of M13-cpHaloΔ9mer and 9mer-CaM linker length: All combinations of split integrators composed of M13-(GGS)1-3-cpHaloΔ9mer and 9mer-(GGS)1-3-CaM were tested in FP kinetic assays in the presence of calcium or EGTA. [Figure 7] Figure 1. General concept of intramolecular calcium integrator based on split-HaloTag7 (CaProLa): (A): Schematic of the basic structure of intramolecular calcium integrator CaProLa (N-terminus to C-terminus). (B): The two parts of the split calcium integrator are joined together, connecting CaM and M13 with a linker domain to form a single-chain sensor. (C): Modeled structure of CaProLa: The crystal structures of HaloTag7 (PDB 4KAF) and the CaM-M13 domains of GCaMP3 (PDB 3SG3) have been modified and arranged to resemble the CaProLa architecture. [Figure 8]Figure 1 shows the performance of CaProLa constructs using different M13-CaM linkers: FP kinetics of different CaProLa constructs and split integrator under four conditions: in the presence of calcium (red), in the absence of calcium (purple), in the absence of calcium but with calcium spike after 1 hour (cyan), in the presence of calcium followed by EGTA spike to stop the reaction followed by calcium spike to restart the reaction (green). Small arrows indicate EGTA and calcium spikes. The curve in the presence of calcium (red) was fitted to a second-order kinetic equation to determine the rate constant. All experiments were performed in triplicate. GGTGGS: SEQ ID NO: 027. [Figure 9] Figure 1 shows calcium responsiveness of CaProLa constructs with different M13-CaM linkers: (A) Labeling reactions of CaProLa constructs were observed via FP at free calcium concentrations ranging from 0 nM to 39 μM. Curves were fitted to a second-order kinetic equation and initial reaction rates were calculated from this fit. (B) Initial rates were plotted versus free calcium concentration (0 nM calcium adjusted to 0.01 nM and displayed on a logarithmic scale) and a four-parameter logistic model was fitted to the data to determine the EC50 of each construct. These experiments concern the intramolecular ΔcpHalo linker between effector moiety 1 and effector moiety 2, and not the intermodular linker between sensor and effector. All experiments were performed in triplicate and error bars and annotated uncertainties represent standard deviations. GGSGGT: SEQ ID NO: 028. [Figure 10] Figure 5 shows the EC50 of second generation CaProLa constructs: (A) Labeling responses of CaProLa 2.1-2.3 were obtained via FP at free calcium concentrations ranging from 10 nM to 3.2 µM. Curves were fitted to a second-order kinetic equation and initial reaction rates were calculated from this fit. (B) Initial rates were plotted against free calcium concentration and a four parameter logistic model was fitted to the data to determine the EC50 for each construct. All experiments were performed in triplicate and error bars and annotated uncertainties represent standard deviations. [Figure 11] Figure 1. Performance of two-generation CaProLa constructs: Fluorescence polarization kinetic assays using Halo-TMR for CaProLa 2.1-2.3 were performed under four conditions: in the presence of calcium (red), in the absence of calcium (purple), in the absence of calcium but with calcium spiked after 1 h (cyan), and in the presence of calcium with EGTA spiked to stop the reaction followed by calcium spike (green). Small arrows indicate EGTA and calcium spikes. The curve in the presence of calcium (red) was fitted to a second-order kinetic equation to determine the rate constant. All experiments were performed in triplicate. [Figure 12] Calcium-dependent labeling of CaProLa2.2 assayed via gel kinetics: (A) CaProLa2.2 was incubated with Halo-CPY in the presence of calcium or EGTA. Samples were taken after various incubation times and analyzed by SDS-PAGE followed by in-gel fluorescence scanning (gels are false-colored to highlight faint bands). (B) Fluorescence intensity was quantified and normalized to fully labeled HaloTag7. [Figure 13] Figure 1 shows a model system for testing split-HaloTag performance: Left: Scheme illustrating the FKBP / FRB-based model system. cpHaloΔ and 9mer peptides were fused to FKBP and FRB, respectively, and the complementation and activity of this split will depend on their rapamycin-dependent interaction. Right: Labeling kinetics of the model system was followed by fluorescence polarization assay in the presence or absence of rapamycin (Rap). Halo-TMR was used as a substrate and each experiment was performed in triplicate. [Figure 14] Figure 1 shows the effect of different flexible linkers in the FKBP / FRB model system: The labeling kinetics of split-HaloTags fused to FKBP and FRB using different flexible linkers was observed via fluorescence polarization assay in the presence or absence of rapamycin. Halo-TMR was used as a substrate and each experiment was performed in triplicate. [Figure 15] A schematic diagram of the split system is shown (the sensor module is split into two peptide chains, each connected to a part of the effector system). [Figure 16] Figure 1 shows labeling kinetics study of ALA scan for 10mer complementary peptide of cpHaloΔ. [Figure 17] Labeling kinetics of dGluProLa followed by fluorescence polarization. Technical triplicates ± standard error (SEM). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0008] Summary of the Invention The present invention relates to fusion proteins comprising a circularly permuted haloalkane transferase split into two parts that are catalytically active when the sensor polypeptide modules are in spatial proximity, but are otherwise catalytically inactive.

[0009] In particular, the present invention provides - an N-terminal first effector sequence portion characterized by SEQ ID NO: 002 or a sequence that is ≥ 90% identical to SEQ ID NO: 002, a C-terminal first effector sequence portion characterized by SEQ ID NO: 003 or a sequence that is ≥ 90% identical to SEQ ID NO: 003, an internal cpHalo linker consisting of 10 to 35 amino acids, which connects the C-terminus of the N-terminal first effector sequence portion to the N-terminus of the C-terminal first effector sequence portion; The present invention relates to a modular polypeptide system comprising a first partial effector sequence connected to a sensor module sequence, the first partial effector sequence comprising: the sensor module sequence is connected to a second partial effector sequence comprising or consisting essentially of a sequence selected from SEQ ID NOs: 006 (PEP1) and 007 (PEP2), or a sequence ≧75% identical to SEQ ID NO: 007 (PEP2); the first and second partial effector sequences together constitute a circularly permuted haloalkane dehalogenase, which when brought into close proximity with one another can result in covalent attachment of a haloalkane moiety, and the sensor module comprises: a. a single sensor polypeptide capable of undergoing a conformational change from a first conformation to a second conformation in response to the presence or concentration of an analyte compound; When the first partial effector sequence is bound to the C-terminus of the sensor module sequence (the single sensor polypeptide) and the second partial effector sequence is bound to the N-terminus of the sensor module sequence (the single sensor polypeptide), in the first conformation, the first and second partial effector sequences are in close proximity (which results in the first and second partial effector sequences constituting a catalytically active entity); and in the second conformation, the first and second partial effector sequences are not in close proximity (which results in the first and second partial effector sequences constituting a catalytically inactive entity); the single sensor polypeptide, and b. a sensor polypeptide pair comprising a first sensor polypeptide and a second sensor polypeptide, wherein the first sensor polypeptide is covalently bound to the first partial effector sequence via a peptide bond, and the second sensor polypeptide is covalently bound to the second partial effector sequence; is selected from the first sensor polypeptide and the second sensor polypeptide are capable of specific intermolecular interaction (protein-protein binding); And the first sensor polypeptide and the second sensor polypeptide are part of separate polypeptide chains.

[0010] A second aspect of the present invention relates to a nucleic acid encoding the fusion protein of the present invention. Alternatively, a nucleic acid encoding two parts of a circularly permuted haloalkane transferase (a first partial effector sequence and a second partial effector sequence) is provided. These nucleic acid sequences encoding the first and second partial effectors are useful for creating other fusion proteins that can sense the concentration of an analyte or a protein-protein interaction using as yet unidentified interaction partners or sensor modules.

[0011] Furthermore, the invention provides expression systems, cells, and transgenic non-human animals that contain the fusion proteins of the invention or the encoding nucleic acids. Similarly, kits that provide the nucleic acids and suitable substrate compounds for the rapid construction of transgenic expression constructs are also encompassed by the invention.

[0012] Detailed Description of the Invention Terms and Definitions The term "fluorescent dye" in the present context refers to a small molecule capable of fluorescing in the visible or near infrared spectrum. Examples of fluorescent labels or labels that exhibit visible color include, but are not limited to, fluorescein isothiocyanate (FITC), rhodamine, allophycocyanin (APC), peridinin chlorophyll (PerCP), phycoerythrin (PE), alexa Fluor® (Life Technologies, Carlsbad, CA, USA), dylight fluor® (Thermo Fisher Scientific, Waltham, MA, USA), ATTO Dye (ATTO-TEC GmbH, Siegen, Germany), BODIPY Dye (4,4-difluoro-4-bora-3a,4a-diaza-s-indacene-based dyes), etc.

[0013] Amino acid sequences are given from the amino terminus to the carboxyl terminus. Capital letters in sequence positions refer to the L-amino acids in their one-letter code (Stryer, Biochemistry, 3rd ed., p. 21). Lower case letters in amino acid sequence positions refer to the corresponding D- or (2R)-amino acids.

[0014] J is isoleucine or leucine.

[0015] The term "polypeptide" in the context of this specification refers to a molecule of 50 or more amino acids forming a linear chain in which the amino acids are connected by peptide bonds. The amino acid sequence of a polypeptide may represent the amino acid sequence of an entire protein (as found physiologically) or a fragment thereof.

[0016] The term "peptide" in the context of the present specification relates to a molecule consisting of up to 50 amino acids, in particular 8 to 30 amino acids, more in particular 8 to 15 amino acids, which amino acids form a linear chain connected by peptide bonds.

[0017] In the present context, the terms "sequence identity" and "percentage of sequence identity" refer to values ​​determined by comparing two aligned sequences. Methods of sequence alignment for comparison are well known in the art. Sequence alignment for comparison can be performed by Smith and Waterman's local homology algorithm, Adv. Appl. Math. 2:482 (1981), Needleman and Wunsch's global alignment algorithm, J. Mol. Biol. 48:443 (1970), Pearson and Lipman's search for similarity method, Proc. Nat. Acad. Sci. 85:2444 (1988), or computer implementations of these algorithms, such as, but not limited to, CLUSTAL, GAP, BESTFIT, BLAST, FASTA, and TFASTA. Software for performing BLAST analyses is publicly available from, for example, the National Center for Biotechnology-Information (http: / / blast.ncbi.nlm.nih.gov / ).

[0018] An example of an amino acid sequence comparison is the BLASTP algorithm with default settings: expectation threshold: 10; word size: 3; maximum match in query range: 0; matrix: BLOSUM62; gap cost: presence 11, extension 1; composition adjustment: conditional composition score matrix adjustment. An example of a nucleic acid sequence comparison is the BLASTN algorithm with default settings: expectation threshold: 10; word size: 28; maximum match in query range: 0; match / mismatch score: 1.-2; gap cost: linear. Unless otherwise specified, the sequence identity values ​​described herein refer to values ​​obtained using the BLAST suite of programs (Altschul et al., J. Mol. Biol. Biol. 215: 403-410 (1990)) using the default parameters identified above for protein and nucleic acid comparisons, respectively.

[0019] In the context of this specification, the term "having substantially the same activity" relates to the activity of the effector polypeptide pair, in particular the activity of SEQ ID NO: 004 and SEQ ID NO: 007 (PEP2), i.e. the haloalkane transferase activity. A polypeptide identified as having "substantially the same activity" does not necessarily have to exhibit the same amount of activity as the reference polypeptide, and in the particular case of the present invention, a reduction in the enzyme turnover number relative to the reference peptide cpHalo may indeed be desirable for certain applications. As described below, with a view to distinguishing between polypeptides covered by the present invention and those not covered by it, the inventors have demonstrated that the enzyme turnover number is 10 times higher in a standard assay as described in Example 9, using Halo-CPY as substrate. 2 s -1 M -1 They propose a threshold for activity.

[0020] In cases where the above definition of activity is not applicable, 3 standard deviations above background for haloalkane transferase activity shall be taken as the criterion threshold for "having substantially the same activity". In certain embodiments, at least 5 standard deviations are used as the criterion threshold. In particularly certain embodiments, at least 10 standard deviations are used as the criterion threshold.

[0021] In the present context, the term "amino acid linker" refers to a polypeptide of variable length used to connect two polypeptides to produce a single polypeptide chain. Unless otherwise specified, exemplary embodiments of linkers useful in the practice of the invention defined herein are oligopeptide chains of 1, 2, 3, 4, 5, 10, 20, 30, 40 or 50 amino acids.

[0022] In the present context, the term "Gltl" is an abbreviation for bacterial periplasmic glutamate-binding protein (Uni-Prot-ID: H4UFY3).

[0023] A first aspect of the present invention relates to a modular analyte sensor polypeptide system (also referred to herein as modular polypeptide) comprising or consisting essentially of a split effector polypeptide sequence having Halotag7 activity, reconstituted by a sensor polypeptide module.

[0024] The modular analyte sensor polypeptide system comprises or essentially consists of a first partial effector polypeptide sequence (cpHaloΔ) comprising or consisting essentially of an N-terminal first effector sequence portion characterized by SEQ ID NO:002 or a sequence at least (≧) 90% identical (particularly ≧93%, ≧95%, ≧97% or ≧98% identical) to SEQ ID NO:002, a C-terminal first effector sequence portion characterized by SEQ ID NO:003 or a sequence at least (≧) 90% identical (particularly ≧93%, ≧95%, ≧97% or ≧98% identical) to SEQ ID NO:003, and an internal cpHalo linker of 10 to 35 amino acids.

[0025] The internal cpHalo linker connects the C-terminus of the N-terminal first effector sequence portion to the N-terminus of the C-terminal first effector sequence portion.

[0026] In certain embodiments, the cpHalo linker consists of 12 to 20 amino acids. In certain embodiments, the cpHalo linker consists, in particular, of about 15 amino acids.

[0027] The first partial effector polypeptide sequence is covalently linked via its N-terminus as part of a peptide chain to the sensor module polypeptide sequence, which in turn is linked as part of a peptide chain to a second partial effector peptide sequence that complements the sequence of cpHaloΔ and reconstitutes haloalkane transferase activity, comprising or consisting essentially of a sequence selected from SEQ ID NO: 006 (PEP1), and 007 (PEP2), or a sequence at least (≧) 75% identical (particularly particularly ≧80%, ≧85%, ≧90%, or ≧95% identical) to SEQ ID NO: 007 (PEP2).

[0028] The first and second partial effector sequences together comprise a circularly permuted haloalkane dehalogenase and are capable of resulting in the covalent attachment of a halogenalkane moiety to the first effector sequence when in proximity to one another.

[0029] Certain variations of the modular analyte sensor polypeptide system of the present invention may be designed to exhibit significantly less activity than the examples provided herein. Native Halotag7 and its circularly permuted forms are highly active, and for some applications lower / slower activity may be desirable. One example is recording neuronal activity over long periods of time, where a highly active system may tend to quickly oversaturate the system and prevent recording of events unfolding over long periods of time.

[0030] As defined herein, the threshold for a combination of split effector module and sensor module sequences is 10 in a standard assay such as that shown in Example 9. 2 s -1 M -1 A minimum activity of is provided.

[0031] In a particular embodiment, a combination of a split effector module consisting of a first effector sequence that is ≧90% identical to SEQ ID NO:004 or to a construct consisting of SEQ ID NO:002 and SEQ ID NO:003 linked by a linker, and a second partial effector sequence, is characterized by a value of at least 3 standard deviations (≧3Σ) above the background signal, in particular a value of ≧5Σ or even ≧10Σ above the background signal, when a variant of SEQ ID NO:004 is in spatial proximity to SEQ ID NO:007 (PEP2).

[0032] For the purposes of calculating sequence identity, the contributions of SEQ ID NO:002 and SEQ ID NO:003 should be weighed, although it will be understood that the linker molecule joining the two sequences can be varied substantially without affecting the function of the resulting construct.

[0033] In a particular embodiment, a combination of a split effector module consisting of a first effector sequence that is ≧90% identical to SEQ ID NO:004 or to a construct consisting of SEQ ID NO:002 and SEQ ID NO:003 linked by a linker, and a second partial effector sequence, is characterized by an activity of at least 0.5%, in particular ≧1 or ≧2%, of SEQ ID NO:004 when in spatial proximity to SEQ ID NO:007 (PEP2).

[0034] In certain embodiments, a combination of a split effector module consisting of a first effector sequence that is ≧90% identical to SEQ ID NO:004 or to a construct consisting of SEQ ID NO:002 and SEQ ID NO:003 linked by a linker, and a second partial effector sequence, is characterized by at least 5% of the activity of SEQ ID NO:004 when SEQ ID NO:004 is in spatial proximity to SEQ ID NO:007 (PEP2).

[0035] In certain embodiments, a combination of a split effector module consisting of a first effector sequence that is ≧90% identical to SEQ ID NO:004 or to a construct consisting of SEQ ID NO:002 and SEQ ID NO:003 linked by a linker, and a second partial effector sequence, is characterized by at least 15%, 25%, 40%, or even 50% of the activity of SEQ ID NO:004 when in spatial proximity to SEQ ID NO:007 (PEP2).

[0036] The sensor module (also referred to herein as a sensor module sequence) is selected from a single sensor polypeptide and a sensor polypeptide pair.

[0037] The sensor module may be a single sensor polypeptide that can undergo a conformational change from a first conformation to a second conformation in response to the presence or concentration of an analyte compound adjacent to the sensor module sequence. The conformational change may also be affected by the absence of the analyte or its removal from the environment. When a first partial effector sequence is bound to the C-terminus of the sensor module (single sensor polypeptide) and a second partial effector sequence is bound to the N-terminus of the sensor module (single sensor polypeptide), in the first conformation, the first and second partial effector sequences are in close proximity, in other words, their proximity results in the first and second partial effector sequences constituting a catalytically active entity.

[0038] In contrast, in the second conformation, when the first partial effector sequence is bound to the C-terminus of the sensor module and the second partial effector sequence is bound to the N-terminus of the sensor module sequence, the first and second partial effector sequences are not in close proximity, thus resulting in the first and second partial effector sequences constituting a catalytically inactive entity.

[0039] The C-terminus of the single sensor polypeptide is covalently linked to the N-terminus of the first partial effector sequence via a direct peptide bond or via a linker peptide, and the N-terminus of the single sensor polypeptide is covalently linked to the C-terminus of the second partial effector sequence.

[0040] Alternatively, a sensor module can be a sensor polypeptide pair comprising a first sensor polypeptide and a second sensor polypeptide, where the C-terminus of the first sensor polypeptide is covalently linked to the N-terminus of a first partial effector sequence and the N-terminus of the second sensor polypeptide is covalently linked to the C-terminus of a second partial effector sequence, either via a direct peptide bond or via a linker peptide, and the first sensor polypeptide and the second sensor polypeptide are capable of specific molecular interaction (protein-protein binding). In this alternative, the first and second sensor polypeptides are part of separate polypeptide chains.

[0041] The second partial effector sequence complements the first partial effector sequence when adjacent. We found that SEQ ID NO: 006 (PEP1) and 007 (PEP2), which differ in a C-terminal threonine residue, are the most effective at doing so. This does not preclude further future development of the peptides to increase the affinity of these partial sequences for each other or for other purposes. One extra threonine in the PEP2 sequence significantly increases the reaction rate in combination with the calmodulin / M13 sensor system, but has little effect when used in combination with the FKPB / FRB sensor system.

[0042] Those skilled in the art will understand that variants of the second partial effector sequence may also be tolerated, depending on the contribution of the individual positions to the structure. Ala145, Arg146, Thr148, Phe149, Phe152, and Arg153, known to interact with proteins, are unlikely to be replaceable with residues of similar chemical properties (or even less similar). Solvent-exposed amino acids (i.e., Glu147, Gln150, Ala151, and Thr154) would a priori be less important to keep exactly the same. In the inventor's hands, mutational studies confirmed these opinions. Position Ala150 turned out to be tolerant to modification, and positions Arg146 and Thr148 turned out to be important for integrator function (in the context of calcium signal integration).

[0043] Since peptides must fold into an alpha-helical structure, AAs can be hierarchically organized using a table of alpha-helical-prone AAs, such a table can be found in Pace et al., Biophysical Journal, 75, 422-427, doi:10.1016 / s0006-3495(98)77529-0.

[0044] In certain embodiments, the first partial effector polypeptide sequence and the second partial effector sequence, when adjacent to each other, together have at least 0.5%, in particular ≧1% or ≧2% of the biological activity of SEQ ID NO: 001. In certain embodiments, the first partial effector polypeptide sequence and the second partial effector sequence, when adjacent to each other, have at least 10% or more of the biological activity of SEQ ID NO: 001 in a standard assay, as shown in Example 9. 2 s -1 M -1 It has activity.

[0045] The activity of SEQ ID NO:001 for purposes of defining sequences encompassed by the preceding definition and other such definitions contained herein is the performance of the sequence in the fluorescence polarization assay of Example 9. Unless otherwise stated, said activity is measured according to the procedure given in Example 9 using Halo-CPY (see Table of Substrates below) for purposes of comparison of biological activity.

[0046] In certain embodiments, the internal cpHalo linker comprises or consists of the amino acids G, A, J, S, T, P, C, V, M, in particular said cpHalo linker comprises or consists of the amino acids G, S, A, T.

[0047] In certain embodiments, the cpHalo linker is (GGΣ) n where n is an integer and n≧3 (particularly n is 4, 5, 6, 7 or 8), and Σ is selected from S and T.

[0048] In certain embodiments, the cpHalo linker is (GGS) n where n is an integer and n >= 3 (particularly, n is 4, 5, 6, 7 or 8).

[0049] In certain embodiments, the cpHalo linker is (GGT) n where n is an integer and n >= 3 (particularly, n is 4, 5, 6, 7 or 8).

[0050] In certain embodiments, the cpHalo linker is (GΣG) n Or (GGΣ) n where n is an integer and n≧3 (particularly, n is 4, 5, 6, 7, or 8), and each Σ is independently selected from S and T.

[0051] In certain embodiments, the cpHalo linker is (ΓΓΣ) n wherein n is an integer and n > 3 (particularly, n is 4, 5, 6, 7, or 8), and each Γ, independently of any other Γ, is selected from A, G, and V, and each Σ, independently, is selected from S and T.

[0052] Regarding the length and sequence composition of the cpHalo linker, our results indicate that any linker with a length equivalent to 10, optimally 12 amino acids, would be expected to function, with the exception of linkers whose predicted structure would be expected to hinder the solubility of the resulting protein. Although we decided not to investigate linkers longer than 25 amino acids, there is no reason why linkers of such length should not be expected to function.

[0053] The main considerations in selecting the linker sequence were solubility and flexibility. The actual cpHalo linker sequence selected for the examples disclosed herein is GGTGGSGGTGGSGGS (SEQ ID NO: 005), but those skilled in the art can easily vary this sequence in composition and length based on the teachings herein and available knowledge on linker design, as exemplified in Chen et al., Advanced Drug Delivery Reviews 65 (2013), 1357-1369, and Evers et al., Biochemistry 2006, 45, 13183-13192.

[0054] In a particular embodiment, the first partial effector polypeptide sequence comprises or essentially consists of SEQ ID NO:004, or a sequence at least (≧) 90% identical (particularly ≧93%, ≧95%, ≧97% or ≧98% identical) to SEQ ID NO:004, and said first and second partial effector sequences together are characterized by at least 1% of the activity of SEQ ID NO:004 in spatial proximity to SEQ ID NO:007 (PEP2).

[0055] In certain embodiments, the first partial effector polypeptide sequence comprises or essentially consists of a sequence at least (≧) 90% identical (particularly ≧93%, ≧95%, ≧97% or ≧98% identical) to SEQ ID NO:004, and said first and second partial effector sequences together are characterized by ≧5%, 10%, 17% or 33% of the activity of SEQ ID NO:004 in spatial proximity to SEQ ID NO:007 (PEP2).

[0056] The sensor and effector modules can be directly connected to each other, but to allow some adjustment in structure, the skilled artisan will recognize that linker sequences can be used to connect the modules.

[0057] In certain embodiments, a first partial effector polypeptide sequence is connected to the sensor module polypeptide sequence (also referred to herein as the sensor module sequence) by a first inter-modular linker peptide sequence, and / or a second partial effector polypeptide sequence is connected to the sensor module polypeptide sequence by a second inter-modular linker polypeptide sequence.

[0058] The inventors have discovered that the GGΨ complex is a cyclic amide complex, wherein n is an integer and n≦4 (particularly n is 1, 2 or 3) and Ψ is selected from G, S and T. n We have now found a first particularly useful inter-modular linker sequence, characterized in that:

[0059] Similarly, we have discovered compounds that are suitable for use in the present invention, where n is an integer and n≦4 (particularly n is 1, 2 or 3) and Ψ is selected from G, S and T, (GGΨ) n We have now found a particularly useful second inter-modular linker sequence which is characterized by:

[0060] In certain embodiments, the first and / or second intermodule linker may be one or two amino acid linkers GΨ, ΨG, ΨΨ, G, or Ψ alone, where Ψ is selected from G, S, and T. Alanine is an example of an amino acid that can substitute for one of G, S, and T in the above sequences.

[0061] Particular importance should be placed on the absence of background labeling and significant signal above background after reconstitution in the presence of the analyte of interest. Depending on the sensor module, a person skilled in the art could vary the intermodule linker sequence and test which length gives the best results for a given combination of modules. In terms of sequence composition, but not length, the alternative sequences presented as examples for the cpHalo linker above also apply to the intermodule linker.

[0062] In certain embodiments, the sensor module sequence is a single sensor polypeptide consisting of a first partial sensor sequence at the N-terminus and a second sensor sequence at the C-terminus connected by a sensor linker sequence.

[0063] In certain embodiments, the sensor module sequence is an oligoproline sequence, particularly P n The present invention relates to a single sensor polypeptide that consists of a first partial sensor sequence at the N-terminus and a second partial sensor sequence at the C-terminus connected by a rigid sensor linker sequence, such as can be achieved by sequencing.

[0064] In certain embodiments, the sensor linker sequence is a polyproline sequence, such as, but not limited to, the polyproline sequence exemplified in the previous section (GGΨ) n(n is an integer, n is selected from 1, 2, 3, and Ψ is selected from G, S, and T). Similarly, the inserted linker may have a short length, such as GΨ, ΨG, ΨΨ, G, or Ψ alone. An example that has worked in our hands is a 15 proline stretch followed by two GGS repeats, again followed by 15 prolines. Such a design can facilitate a hinge in the spring formed by the prolines. We have observed that linkers (mostly rigid) offer certain advantages, but the linker sequence can be varied over a wide range of sequences without compromising the activity of the final construct. Again, it must be remembered that in certain applications, a low activity that is still clearly distinguishable from background noise may actually be preferable to a highly active construct. Brun et al. (J Am Chem Soc 2011, 133(40)16235-16242) teach a variety of linkers that are applicable to the present invention. See again Chen et al., (ibid.) and Evers et al., (ibid.), supra.

[0065] In certain embodiments, the first partial sensor sequence and the second partial sensor sequence are selected from a calmodulin binding peptide and a calmodulin polypeptide.

[0066] In particularly specific embodiments, the first partial sensor sequence is a calmodulin polypeptide and the second partial sensor sequence is a calmodulin binding peptide.

[0067] A large number of different calmodulin mutants and binding peptides exist, as described inter alia in: Zhao et al., Science 2011 vol 333(6051)1888-91 doi:10.1126 / science.1208592; Wu et al., 2013 ACS Chemical Neuroscience vol 4(6)963-972 doi:10.1021 / cn400012b; Horikawa et al., 2010 NatMethods 2010 Sep;7(9):729-32 doi:10.1038 / nMeth.1488; Chen et al., Nature 2013 Jul 18;499(7458):295-300, doi:10.1038 / nature12354; Moeyaert et al., Nat Commun. 2018 Oct 25;9(1):4440 doi:10.1038 / s41467-018-06935-2;Dana et al., 2018 bioRxiv 434589;Gao et al., 2015 doi:10.1038 / nn.4016;Lee et al., 2017 doi:10.1038 / nbt.3902;Minderer et al., 2012 doi:10.1113 / jphysiol.2011.219014;De Juan-Sanz et al., 2017 doi:10.1016 / j.neuron.2017.01.010;Ding et al., 2015 doi:10.1038 / nmeth.3261 and Akerboom et al., 2011, doi:10.1523 / JNEUROSCI.2601-12.2012.

[0068] In certain embodiments, the calmodulin polypeptide is or comprises SEQ ID NO: 009 (CaM), or a sequence that is at least 90% identical to SEQ ID NO: 009 (CaM) and has substantially the same biological activity, and the calmodulin binding peptide is or comprises SEQ ID NO: 008 (M13).

[0069] In a particular embodiment, the sensor module is composed of a sensor polypeptide pair comprising a first sensor peptide that is or comprises a calmodulin binding peptide exemplified by SEQ ID NO: 008 (M13) and a second sensor polypeptide that is or comprises a calmodulin polypeptide, in particular SEQ ID NO: 009 (CaM), or a sequence at least 90% identical to SEQ ID NO: 009 (CaM) and having substantially the same activity, wherein the first sensor peptide is covalently linked to a first partial effector sequence via a peptide bond and the second sensor polypeptide is covalently linked to a second partial effector sequence, and the first and second sensor polypeptides are part of separate polypeptide chains.

[0070] In certain embodiments, the calmodulin-binding peptide is selected from any of the calmodulin-binding peptides known in the art. Longer variants that function in the same way have been reported, see references cited above.

[0071] In particularly specific embodiments, the first partial effector sequence is connected to the C-terminus of the first sensor peptide by a first inter-modular linker sequence having 2-6 amino acids and / or the second partial effector sequence is connected to the N-terminus of the second sensor polypeptide by a second inter-modular linker sequence having 2-6 amino acids.

[0072] In more particularly specific embodiments, the first inter-modular linker sequence and the second inter-modular linker sequence are dipeptides or tripeptides, the amino acid components of which are each independently selected from G, S and T residues.

[0073] In a particular embodiment, the modular polypeptide is characterized by a first polypeptide sequence consisting of or comprising SEQ ID NO:010 (SPLT1) or a sequence at least 90% identical to SEQ ID NO:010 (SPLT1), and a second polypeptide sequence which is SEQ ID NO:011 (SPLT2) or a sequence at least 90% identical to SEQ ID NO:011 (SPLT2), said first and second polypeptide sequences together having at least 0.5%, in particular ≧1% or ≧2% of the activity of the combination of SEQ ID NO:010 (SPLT1) and SEQ ID NO:011 (SPLT2). In a particular embodiment, the sensor module comprises from the N-terminus to the C-terminus: Calmodulin polypeptides, in particular SEQ ID NO: 009 (CaM), or a sequence that is at least 90% identical to SEQ ID NO: 009 (CaM) and has substantially the same activity; Peptide sensor linker sequences, particularly polyproline-type rigid helices, more particularly P, where n is an integer selected from 15 to 35 and is optionally flanked by 1 to 4 amino acids. n a proline polypeptide or a polyproline sequence interrupted by a short flexible stretch of 1-10 residues selected from G, T, S, A; Calmodulin-binding peptide (second partial sensor sequence), in particular a sequence comprising or consisting of SEQ ID NO: 008 (M13) It is composed of a single sensor peptide comprising:

[0074] In a particular embodiment, the modular polypeptide of the invention comprises or consists of a sequence selected from SEQ ID NO: 013 (CONF1) and SEQ ID NO: 014 (CONF2), or a sequence at least 90% identical to SEQ ID NO: 014 (CONF2) or SEQ ID NO: 013 (CONF1) having at least 0.5%, in particular ≧1% or ≧2% of the activity of SEQ ID NO: 013 (CONF1).

[0075] In certain embodiments, the sensor module array comprises or consists essentially of: a. the N-terminal portion of a glutamate binding protein, in particular the first sensor polypeptide is or comprises SEQ ID NO: 020 (GLT1), or a sequence that is at least 90% identical to SEQ ID NO: 020 (GLT1), and a C-terminal portion of a glutamate binding protein, in particular wherein the second sensor polypeptide is or comprises SEQ ID NO: 021 (GLT2), or a sequence which is at least 90% identical to SEQ ID NO: 021 (GLT2) and has substantially the same biological activity, in particular derived from a bacterial periplasmic glutamate binding protein, more in particular Gltl; wherein the combination of the first sensor polypeptide and the second sensor polypeptide has substantially the same biological activity as the combination of SEQ ID NO:020 (GLT1) and SEQ ID NO:021 (GLT2); or b. A sequence that is at least (≧) 90% identical to a construct consisting of SEQ ID NO: 020 (GLT1) linked to SEQ ID NO: 021 (GLT2) by a polypeptide linker, in particular wherein the sequence of the sensor module is or comprises SEQ ID NO: 022 (GLT3), or a sequence that is at least 90% identical to SEQ ID NO: 022 (GLT3) and has substantially the same biological activity.

[0076] In a particular embodiment, the modular polypeptide is characterized by a first polypeptide sequence consisting of or comprising SEQ ID NO: 023 (GLTIND1), or a sequence that is at least 90% identical to SEQ ID NO: 023 (GLTIND1), and a second polypeptide sequence that is SEQ ID NO: 024 (GLTIND2) or a sequence that is at least 90% identical to SEQ ID NO: 024 (GLTIND2), said first and second polypeptide sequences together having at least 0.5%, in particular ≧1% or ≧2% of the activity of SEQ ID NO: 025 (GLTIND3).

[0077] In certain embodiments, the sensor module is comprised of a sensor polypeptide pair comprising: a first sensor polypeptide which is or comprises an FKBP12 polypeptide, in particular the FKBP12 polypeptide is or comprises SEQ ID NO: 015 (FKBP) or a sequence which is at least 90% identical to SEQ ID NO: 015 (FKBP) and has essentially the same biological activity; and a second sensor polypeptide which is or comprises an FRB peptide, in particular the FRB peptide is or comprises SEQ ID NO: 016 (FRB) or a sequence which is at least 90% identical to SEQ ID NO: 016 (FRB) and has essentially the same biological activity; wherein the first sensor polypeptide is covalently linked to a first partial effector sequence via a peptide bond, and the second sensor polypeptide is covalently linked to a second partial effector sequence, and the first sensor polypeptide and the second sensor polypeptide are part of separate polypeptide chains.

[0078] In particularly specific embodiments, the first partial effector sequence is connected to the C-terminus of the first sensor polypeptide by a first inter-modular linker sequence having 2-9 amino acids and / or the second partial effector sequence is connected to the N-terminus of the second sensor polypeptide by a second inter-modular linker sequence having 2-9 amino acids.

[0079] In a more particularly specific embodiment, the first inter-modular linker sequence and the second inter-modular linker sequence are tripeptides, the amino acid components of which are each independently selected from G, S and T residues.

[0080] In a particular embodiment, the modular polypeptide is characterized by a first polypeptide sequence consisting of or comprising SEQ ID NO: 017 (RAPIND1) or a sequence at least 90% identical to SEQ ID NO: 017 (RAPIND1), and a second polypeptide sequence selected from SEQ ID NO: 018 (RAPIND2) and SEQ ID NO: 019 (RAPIND3) or a sequence at least 90% identical to SEQ ID NO: 018 (RAPIND2), said first and second polypeptide sequences together having at least 0.5%, in particular ≧1%, or ≧2% of the combined activity of SEQ ID NO: 017 (RAPIND1) and SEQ ID NO: 018 (RAPIND2). In a particularly particular embodiment, the first and second polypeptide sequences together have 5%, 10%, 15%, 20% or 33% of the combined activity of SEQ ID NO: 017 (RAPIND1) and SEQ ID NO: 018 (RAPIND2).

[0081] Another aspect of the present invention relates to a nucleic acid sequence or sequences encoding a modular analyte sensor polypeptide according to the present invention, as described in any of the aspects, embodiments or examples herein.

[0082] The present invention can also be embodied by a combination of nucleic acid sequences including: - a first nucleic acid sequence encoding a first partial effector polypeptide sequence, said encoded first partial effector sequence comprising, from N-terminus to C-terminus: SEQ ID NO: 002 or a sequence at least (≧) 90% identical (in particular ≧93%, ≧95%, ≧97% or ≧98% identical) to SEQ ID NO: 002, - a polypeptide linker sequence having 10 to 35 (particularly about 15) amino acids, more particularly 12 to 20 amino acids, selected from G, A, J, S, T; SEQ ID NO: 003 or a sequence at least (≧) 90% identical (in particular ≧93%, ≧95%, ≧97% or ≧98% identical) to SEQ ID NO: 003, the first nucleic acid sequence comprising: - a second nucleic acid sequence encoding a second partial effector peptide sequence, characterized by a sequence of SEQ ID NO: 006 (PEP1), SEQ ID NO: 007 (PEP2) or at least (≧) 95% identical (in particular ≧96%, ≧97%, ≧98% or ≧99% identical) to SEQ ID NO: 006 (PEP1), wherein the first and second partial effector polypeptide sequences together comprise a circularly permuted haloalkane dehalogenase and, when in close proximity to one another, are capable of effecting covalent attachment of a halogenalkane moiety. Again, for purposes of defining sequence variants that are considered to be encompassed by the present invention, the threshold combination of split effector modules that can be combined with a sensor module sequence is one that exhibits an activity of 10 or more in a standard assay as described in Example 9. 2 s -1 M -1 It is.

[0083] In certain embodiments, the split effector modules, when combined under conditions favoring spatial proximity as in the above examples and embodiments, exhibit at least 0.5%, in particular ≧1% or ≧2% of the activity of SEQ ID NO: 001. In certain embodiments, the split effector modules, when combined under conditions favoring spatial proximity as in the above examples and embodiments, exhibit at least 10% or more of the activity of SEQ ID NO: 001 in a standard assay as shown in Example 9. 2 s -1 M -1 It has activity.

[0084] The combination can be provided, for example, as a nucleic acid vector for inserting nucleic acid encoding an experimental sensor module, or for fusion to a pair of encoded peptides whose interaction is examined by the complementary activity of the effector module.

[0085] Another aspect of the present invention relates to a nucleic acid expression system comprising a nucleic acid sequence according to the previous aspect or a combination of nucleic acids encoding an effector module pair of sequences as described herein, each nucleic acid sequence being under the control of a promoter sequence.

[0086] Another aspect of the invention relates to an isolated cell or a transgenic non-primate animal comprising a nucleic acid expression system or a nucleic acid sequence as disclosed herein, in particular wherein the promoter is operative in said cell.

[0087] Similarly, the invention may be embodied by a kit comprising a nucleic acid sequence or a nucleic acid expression system as disclosed herein and a Halotag7 substrate, in particular a haloalkane moiety covalently attached to a fluorescent dye.

[0088] The HaloTag system was first published by Los et al. (ACS Chemical Biology 2008 Vo 3(6)373-382). It is reviewed in England et al. Bioconjugate Chemistry 2015,26 975-986. Patent literature showing general aspects of the HaloTag system and substrate molecules useful therein includes U.S. Pat. No. 8,202,700 B2, U.S. Pat. No. 7,867,726, PCT / US2013 / 074756, U.S. Pat. No. 9,927,430 B2, U.S. Pat. No. 10,168,323 B2, and U.S. Pat. No. 2014,322,738, all of which are incorporated herein by reference.

[0089] Exemplary fluorophore substrates that can be used in the present invention include, but are not limited to, the fluorophore substrate compounds shown in the table below:

[0090] [Table 1] JPEG0007599074000002.jpg218153

[0091] Wherever there are alternatives of single separable features, it should be understood that such alternatives can be freely combined to form separate embodiments of the invention disclosed herein.

[0092] The present invention is further illustrated by the following examples and figures, from which further embodiments and advantages can be derived, which are intended to illustrate the invention but not to limit its scope. EXAMPLES

[0093] Example 1: Design of a calcium signal integrator based on split-HaloTag7 HaloTag7 is a self-labeling protein derived from the haloalkane dehydrogenase DhaA of Rhodococcus rhodochrous that specifically reacts with and covalently binds synthetic chloroalkane ligands. A split system was generated in which the original ends of HaloTag7 were connected via a (GGS / T)5 linker and the peptide between the circular permutant (CP) sites cpHalo141-145 (cpHaloTag7 has new ends at positions 141 and 145) and cpHalo156-154 was excised from the HaloTag7 protein. The portion between these positions was excised to generate a splitter consisting of cpHalo156-141 and a short 9-mer peptide from positions 145-153 / 154.

[0094] As the cpHaloΔ9mer-9mer peptide couple showed promising preliminary results, the first version of the calcium integrator was designed by fusing it to the M13 peptide and calmodulin protein, respectively, via GGS linkers. At higher calcium concentrations, calmodulin binds up to four calcium ions, resulting in a large conformational change that significantly increases its affinity for the M13 recognition peptide. The resulting binding of calmodulin to M13 leads to the complementation of cpHaloΔ9mer by the 9mer peptide. When complemented, the enzyme regains activity and is able to react with the fluorescent HaloTag substrate, leaving a permanent mark, thus integrating this signal.

[0095] Example 2: Affinity between cpHaloΔ9mer and 9mer peptides Isothermal titration calorimetry The affinity between cpHalo_9mer and the 9mer peptide was measured by a label-free approach using isothermal titration calorimetry (ITC).

[0096] The 9mer peptide dissolves at approximately 30 mM in activity buffer (HEPES 50 mM pH 7.3-NaCl 50 mM) and forms gels at high concentrations in a strongly temperature-dependent manner. The highest concentration of this peptide was titrated against cpHaloΔ9mer at 0.6 mM (Figure 1). d The experiment was repeated three times with different batches of cpHaloΔ9mer to confirm the K d Values ​​could be calculated from three independent measurements.

[0097] Example 3: Affinity of fluorescent ligands for Halotag7 and cpHaloΔ9mer We decided to perform experiments with several representative fluorescent HaloTag substrates. Replacement of the active site aspartic acid 106 residue with alanine removed the nucleophile responsible for the autolabeling reaction and eliminated the catalytic activity of the protein. Catalytically inactive mutants of HaloTag7 (HaloTag7D106A) and cpHaloΔ9mer (cpHalo_9merD106A) were generated to measure substrate affinity without shifting the equilibrium through the enzymatic reaction.

[0098] Since binding of fluorogenic ligands by HaloTag7 causes a strong increase in fluorescence, the affinity of different fluorogenic substrates for HaloTag7D106A was determined with an FP binding assay (Figure 2), while the affinity of fluorogenic silicon-rhodamine derived substrates was measured by a fluorescence intensity-based binding assay (Figure 3). In both cases, HaloTag7D106A concentrations ranging from 0 µM to 100 µM were titrated against 25 nM of dye. HaloTag7D106A binds to ligands with affinities ranging from 622 + / - 3.7 nM for Halo-Carbopyronine (Halo-CPY) to 172.2 + / - 8.2 µM for Halo-Janelia Fluor635 (Table 1). The observed affinities are in agreement with the labeling kinetics recorded with the different substrates (data not shown). In general, fluorogenic substrates have lower affinity for HaloTag7D106A than non-fluorogenic substrates, with the exception of Halo-Alexa Fluor 488, which features the same range of affinities. Furthermore, within silicon rhodamine derivatives, the presence of an azetidine or 3-fluoroazetidine moiety influences the binding affinity, which is more pronounced compared to the rhodamine structure. The affinity of the fluorescent HaloTag ligands that show the highest affinity for HaloTag7 was tested using cpHaloΔ9merD106A in the FP binding assay and the results were compared to measurements using HaloTag7D106A (Figure 4, Table 1).

[0099] Example 4: Background labeling of cpHaloΔ9mer A low background is required for the protein complementation assay to function well. Therefore, we measured the degree of background labeling of cpHaloΔ9mer with different fluorophores (Halo-TMR, Halo-CPY and Halo-SiR). Therefore, cpHaloΔ9mer was incubated with an excess of different fluorescent ligands.

[0100] The labeling efficiency at 37°C and different time points was determined by gel fluorescence measurements (Figure 5). Very low background labeling was observed with Halo-TMR and Halo-SiR. Halo-CPY showed slightly stronger background labeling, eventually reaching 20% ​​after 24 h. These findings are in agreement with the affinity of the fluorescent ligands (Figure 4). Indeed, Halo-CPY showed the highest affinity for cpHaloΔ9mer, showing some affinity for the ligand at 2 μM, resulting in minimal remaining labeling, considering the experimental setup.

[0101] Example 5: Characterization and optimization of a split-Halotag7-based calcium signal integrator: Linker optimization The initial design of the calcium integrator consists of fusions of the cpHaloΔ9mer with the M13 peptide and the 9mer peptide with calmodulin. Both fusion partners are derived from the calcium-sensing domain of GCaMP6f. Kinetics by fluorescence polarization using Halo-TMR as substrate show no background in the absence of calcium and a second-order rate constant of labeling of 6.7 + / - 0.5 in the presence of calcium. * 10 3 s -1 M -1 It became clear that...

[0102] An optimal linker should help to place the 9mer peptides at a good distance and in the right orientation to complement the cpHaloΔ9mer. Therefore, three mutants of each part of the split were synthesized with various linker lengths (GGS) 1~3) were generated. These constructs allowed us to screen all combinations of linker length for the best combination to work with. We observed that with increasing linker length, both for the M13-cpHaloΔ9mer linker and the 9mer-calmodulin linker, calcium-induced activity was significantly reduced. Interestingly, the single GGS linker initially selected clearly performed best, as it showed the fastest labeling kinetics with calcium and no detectable background after 2.5 h (Figure 6).

[0103] Example 6: Design and testing of intramolecular calcium integrators based on split-Halotag7 We designed a simplified intramolecular calcium integrator called "CaProLa" (calcium-dependent protein label) by fusing the two integrator compartments between calmodulin and M13 using different linker domains (Figure 7). The following calmodulin-M13 linkers were tested on this first generation CaProLa construct: Flexible GGTGGS (SEQ ID NO: 026) linker - (CaProLa1.1) Partly flexible, partly rigid Pro15-(GGS)2-Pro 15 Linker-(CaProLa1.2) Rigid Pro30 Linker - (CaProLa1.3) SNAP tag domain flanked by Pro15 linkers - (CaProLa1.4)

[0104] Each construct and split system was tested in an FP kinetic assay using Halo-TMR as a substrate under four different conditions (Figure 8): In the presence of calcium to determine labeling kinetics and rate constants (positive experiment) Confirmation by addition of EGTA in the absence of calcium to assay background labeling (negative experiment) To demonstrate activity after a long incubation with substrate, in the absence of calcium, but after 1 hour, calcium was suddenly added (spike) (spike experiment) To demonstrate the reversibility of the sensor, the reaction was subsequently stopped with an EGTA surge in the presence of calcium, followed by a calcium surge to restart the reaction (reversibility experiment)

[0105] All mutants tested showed a 3-fold increase in the expression of β-calcium in the presence of calcium. * 10 3 s -1 M -1 ~5 * 10 3 s -1 M -1 Furthermore, induction and reversibility experiments with calcium spikes after 1 h were successful, suggesting that once the 9mer peptide binds to the cpHaloΔ9mer structure, it can unbind (even in an intramolecular system), providing superior dynamics.

[0106] However, differences in background labeling of the sensors were observed. The split integrator showed no detectable background over 2 h, whereas the CaProLa constructs showed varying degrees of background labeling, which correlated with the stiffness of the M13-CaM linker. The smallest background was observed for the Pro 30 Linker and Pro 15 -SNAP-Pro 15 Observed in the domain.

[0107] Example 7: Calcium responsiveness of different CaProLa constructs The calmodulin-M13 pair selected for the first design of CaProLa was derived from GCaMP6f. Depending on the structural context, the responsiveness of the pair to calcium can vary. The calmodulin moiety binds up to four calcium atoms with a very complex allosteric behavior. However, when incorporated into a sensor, a simple titration of the sensor activity against the free calcium concentration gives an EC 50This leads to the identification of

[0108] The calcium dependence of CaProLa constructs with different M13-CaM linkers was examined using calcium-dependent EC 50 The labeling at different calcium concentrations was therefore monitored with an FP kinetics assay. To achieve defined calcium concentrations in the nanomolar range, a K2EGTA-CaEGTA buffer system was used. The calcium-dependent EC 50 The initial reaction rate was determined in order to calculate the EC 50 The EC values ​​are in the low nanomolar range (19 nM to 146 nM) and depend on the stiffness of the M13-CaM linker. Increasing the stiffness of the linker increases the EC 50 For comparison, GCaMP6f, using the same calmodulin-M13 couple, has an EC 50 Shows.

[0109] Example 8: Modulation of calcium responsiveness of CaProLa constructs The resting calcium concentration of neurons is reported to be 50nM to 100nM. As a result, the first generation CaProLa was thought to be too sensitive to calcium to function in neurons. Therefore, the second generation CaProLa was developed to inhibit calcium synthesis, especially in ECs. 50 With increasing amounts of calcium, we aimed to generate different constructs that exhibited different calcium responsiveness.

[0110] The calmodulin-M13 couple has been extensively studied and numerous mutations have been reported and used in sensors to modify calcium responsiveness. Therefore, we decided to base our design on the as yet unpublished versions of the calcium integrator CaMPARI2 deposited with Addgene (Schreiter, E., Addgene plasmids, no. 101061, no. 101062 and no. 101064). These CaMPARI2 mutants have EC values ​​ranging from 110 nM to 825 nM. 50They are annotated with values ​​and are designed for similar uses.

[0111] Three of the modified M13 peptides were implemented into second generation CaProLa constructs (CaProLa2.1-2.3). These constructs were all implemented in CaProLa1.3 (Pro 30 The EC of the new CaProLa construct is based on the M13-CaM linker. 50 Values ​​were determined as described above (Figure 10).

[0112] EC of CaProLa2.1-2.3 50 The values ​​are comparable to those annotated for CaMPARI2 (Table 2). Both CaProLa2.1 and 2.2 have significantly higher EC values ​​than version 1.4. 50 and may be suitable for integrating neuronal calcium waves (500 nM to 10 μM).

[0113] As with the first generation, all new CaProLa constructs were tested for calcium-induced kinetics, reversibility, and background labeling in FP kinetics experiments (Figure 11). From the three constructs tested, only CaProLa2.2 performed as expected, although it was slightly slower (2.3 + / - 0.014) than the first CaProLa generation. * 10 3 s -1 M -1 The labeling reaction was reversible, with no background observed during the 4-hour incubation. CaProLa2.1, and especially 2.3, showed slightly slower kinetics (rate constant of about 1 * 10 3 s -1 M -1 ), and more importantly, was inferior due to higher background labeling. 50 Since all constructs performed well in the measurements, these experiments need to be repeated to rule out experimental error.

[0114] To confirm the results obtained in the FP assay, the CaProLa2.2 construct was then tested in in-gel fluorescence (Figure 12). In-gel fluorescence measurements were performed using Halo-CPY as substrate, showing that the background begins to become significant after 2 hours with this fluorophore. Due to the nature of the more direct readout (FP observing any binding), these results confirmed the successful function of CaProLa2.2 to promote metastasis in the cellular assay.

[0115] Example 9: Fluorescence polarization based assay to test the performance of Halotag7 or circular permutations of Halotag7: Protein Production and Purification Proteins (HaloTag7-cpHalo variants or X-ProLa) fused to purification tags (His-tag and possible Strep-tag) are expressed in Escherichia coli BL21(DE3)-pLysS strain and purified using classical IMAC (and possible Strep Trap affinity chromatography methods). After buffer exchange and concentration, if necessary (in the case of cpHalo variants), the N-terminal His-tag is removed by TEV cleavage and reverse IMAC purification. The buffer is exchanged to an appropriate buffer (e.g., 50 mM NaCl, 50 mM HEPES, pH 7.3). If necessary, the protein can be further purified by size exclusion chromatography in the same buffer.

[0116] Fluorescence polarization assay Labeling kinetics were performed by mixing 100 μL of protein (at 400 nM) with 100 μL of fluorescent HaloTag substrate (i.e. Halo-CPY) (at 100 nM) in a 96-well plate (black - non-binding - flat bottom) in buffer 50 mM NaCl, 50 mM HEPES, pH 7.3, 0.5 mg / ml BSA. The increase in fluorescence polarization is recorded using a microplate reader equipped with appropriate spectral filters / monochromators (TECAN Spark20). The kinetics of cpHalo variants and HaloTag7 are usually very fast, so a plate reader with an internal injector should be used to minimize the lag between mixing and the start of the measurement. However, even with this equipment, it may not be possible to observe reactions that are completed in less than 1 second. In this case, a stopped-flow setup that allows measuring fluorescence polarization at high sampling rates is necessary (e.g. BioLogicSFM). The reduced sensitivity of such instruments requires increased concentrations of fluorescent substrate and protein (ie, 1:1 mix of 1 μM substrate and 10 μM protein).

[0117] In addition, to account for the effects of dilution and evaporation, the fluorescence polarization time course without any protein is always recorded and subtracted from the data. The obtained kinetic data are fitted to a second-order kinetic law (see equation below) to derive the second-order rate constant (k). To estimate the error, experiments should be performed at least in triplicate. To compare different mutants, all assays should be performed with the same concentration and substrate.

[0118]

number

[0119] Overview of X-ProLa mutant assays Fluorescence polarization assays are also used to test the performance of X-ProLa variants. The general procedure is the same as above. However, these constructs are often slower than Halotag7 or its CP variants, so a plate reader assay may be sufficient. Also, in addition to the fluorescent substrate, the respective metabolite / ion / small molecule that activates the sensor must be added. By recording the labeling kinetics with and without metabolites, the signal above background can be measured, and by titrating different metabolite concentrations, the EC of X-ProLa can be determined. 50 The value can be derived (EC 50 is defined as the concentration at which the velocity of the label is half the maximum velocity of the label).

[0120] Example 10: Protein-Protein Assay System We further tested the performance of our split-HaloTag system for labeling protein-protein interactions in a simple model system. We used the strong interaction of the protein FKBP with FRB, which is conditional on the presence of the small molecule drug rapamycin. After fusing the split-HaloTag fragment to FKBP, FRB labeling was observed only in the presence of rapamycin, indicating that our strategy works in a model system (Figure 13). Different flexible linkers (1-3x GGS) were placed between the interacting proteins (FKBP / FRP) and the split-HaloTag fragment to evaluate the effect of linker length and find high-performing linker combinations (Figure 14). All linker combinations gave at least minimal signal to noise, but certain combinations (FKBP-GGSx3-cpHaloΔ and 9mer-GGSx1-FRB) clearly showed superior performance. These assays indicate that the sensor can integrate protein-protein interactions and could potentially be used to record the activity of signaling pathways in vitro or in vivo.

[0121] Table 1: Catalytically inactive mutant Halotag7 D106A and cpHaloΔ9mer D106A K dThe affinity of the fluorescent ligand to the value is given with the standard error resulting from the non-linear regression.

[0122] [Table 2]

[0123] Example 11: Mutation tolerance scanning of complementary peptides Experimental procedure Fluorescence polarization kinetics performed in buffer 50 mM NaCl, 50 mM HEPES, 100 μM EGTA, 0.5 g / l BSA, pH 7.3. Mix 100 μl of 400 nM protein and 100 nM Halo-TMR in buffer in a black flat-bottom 96-well plate equilibrated at 37°C. Inject 100 μl of 10 mM CaCl2 into the buffer to start the reaction. Final concentrations: 200 nM protein, 50 nM Halo-TMR, 5 mM CaCl2. Add background wells with no added protein. Fluorescence polarization readout until a plateau is reached. Subtract background from measurements. Fit second order kinetics to obtain k2 app get.

[0124] result Mutations of the 10-mer peptide that can complement the activity of cpHaloΔ have a direct and large impact on the sequence conservation (%) compared to the native sequence. Therefore, we performed an alanine scan on the peptide in the context of the already optimized CaProLa construct to evaluate the impact of peptide mutations on the overall labeling kinetics at calcium saturation (Figure 16). This alanine scan means that each residue was mutated one by one to alanine, even if alanine was present, and replaced by leucine, which the inventors consider to be the most "property-conserving" in the context of the α-helical structure.

[0125] And comparison of labeling kinetics suggests the following: - Mutation of Ala145 to leucine affects the kinetics of Integrator, which can be explained by the tight hydrophobic packing of this region, and the recalcitrance of leucine could disrupt this packing and reduce the ability of the peptide to fold into an α-helix and / or to interact with substrates. Mutation of Arg146, Glu147, and Thr148 to alanine did not adversely affect Integrator function, and we hypothesize that at this position the ability to form an α-helix is ​​only essential. - Mutation of Phe149 and Gln150 dramatically reduces the kinetics of the Integrator, especially in the case of the phenylalanine involved in the hydrophobic heart of the substrate regulatory site, which is more surface exposed and likely caps a region that aids in proper folding of the peptide. Mutation of A151 to -leucine unexpectedly resulted in an increase in the labeling rate (approximately 3-fold) compared to the parent protein. Therefore, we further investigated mutations at this position and assessed that while a methionine mutation performed comparably to the parent protein, all other tested modifications (Cys / Phe / Ile / Thr / Val) were detrimental to activity. - Mutation of Phe152 and Arg153 results in the loss of the protein's ability to be labeled. Phe152 is part of the hydrophobic core of the protein active site, while Arg153 interacts with several surrounding residues, most likely both of which are important for proper α-helical folding of the peptide. Mutation of -Thr154 also leads to a decrease in the rate of protein labeling, and this residue appears to fix the peptide in the proper orientation by interacting with residues in the adjacent α-helix.

[0126] In summary, Ala145, Phe149, Gln150, Phe152, and Arg153 appear to be less amenable to modification in the context of the CaProLa sensor, whereas modifications of Arg146, Glu147, and Thr148 are less problematic. Finally, modification of A151 may still lead to increased activity, although this is highly dependent on the nature of the modification.

[0127] Example 12: Development of a glutamate integrator based on cpHaloΔ / H-peptide Experimental procedure Fluorescence polarization kinetic experiments were performed in black flat-bottom 96-well plates at 37°C or 22°C. The buffer composition was 50 mM NaCl, 50 mM HEPES, 0.5 g / L BSA, pH 7.3. 150 μL of 400 nM protein and 2x final concentration of glutamate were equilibrated for 30 min and 100 μL of Halo-CPY was injected into the buffer to start the reaction. Final concentrations of reagents were 200 nM protein and 50 nM Halo-CPY. Fluorescence polarization was read until the readings reached a plateau. Curves were fitted to a monoexponential function in Prism 8 (GraphPad). Saturation of glutamate is observed at 1 mM.

[0128] result We have successfully generated an integrator for glutamate (GluProLa: glutamate-dependent protein label), the major excitatory neurotransmitter in the mammalian central nervous system (CNS). GluProLa is designed around the architecture of iGluSnFR, an existing real-time intensity measurement sensor of glutamate. iGluSnFR is derived from the bacterial periplasmic glutamate-binding protein Gltl. Insertion of a circularly permuted green fluorescent protein (cpGFP) into the flexible hinge of Gltl yielded a green fluorescent sensor that responded to changes in glutamate concentration with an increase in fluorescence intensity. Because the N- and C-termini of Gltl are on the same face of Gltl, and mechanistic studies of iGluSnFR suggest that these positions should show glutamate-binding-dependent changes in relative distance and / or orientation, we reasoned that these were suitable sites for fusion to H-peptide and cpHaloΔ. Therefore, we created a GluProLa construct in which H-peptide (SEQ ID NO: 007 (PEP2)) is linked to the N-terminus of iGluSnFR and cpHaloΔ (SEQ ID NO: 004) is linked to the C-terminus. We cloned and purified a small family of constructs with flexible GGTGGS (SEQ ID NO: 026) and / or Pro10 linkers between H-peptide and iGluSnFR and between iGluSnFR and cpHaloΔ. All constructs showed labeling in the presence of glutamate and fluorescent HaloTag substrate as determined by in vitro fluorescence deflection assay (e.g., FIG. 17). We found that the relative labeling kinetics in the presence or absence of glutamate could be tuned by varying the linker composition. Constructs with GGTGGS (SEQ ID NO: 026) sequences at both the H-peptide / iGluSnFR and iGluSnFR / cpHaloΔ positions showed the lowest nonspecific labeling rates. The present inventors introduced a point mutation (G67A) into cpGFP.This mutation prevents chromophore formation in wtGFP, and gGluProLa (g stands for green) mutants with a G67A mutation (called dGluProLas for dark color) were also found to be non-fluorescent. The GluProLa mutants showed labeling that was dependent on glutamate concentration. The dGluProLa mutants showed slower labeling kinetics than the parent gGluProLa sequence, while maintaining the same affinity for glutamate. The best constructs represent a 15-fold difference in terms of labeling kinetics (i.e. activity) (Figure 17).

[0129] Table 2: Overview of second generation CaProLa constructs using CaM-M13 mutants and ECs reported for CaMPARI2. 50 values, and EC measured for CaProLa 50 [Table 3]

[0130] array Halotag7 (see GenBank AQS79242); the cp version employed in constructing the present invention does not include the C-terminal 27 amino acids of this sequence.

[0131] [Table 4] JPEG0007599074000007.jpg205153JPEG0007599074000008.jpg216153JPEG0007599074000009.jpg220153JPEG0007599074000010.jpg103153

Claims

1. 1. A modular polypeptide comprising: a first partial effector sequence, - an N-terminal first effector sequence portion characterized by SEQ ID NO: 002 or a sequence at least 90% identical to SEQ ID NO: 002, a C-terminal first effector sequence portion characterized by SEQ ID NO: 003 or a sequence at least 90% identical to SEQ ID NO: 003, an internal cpHalo linker consisting of sequence SEQ ID NO: 005, said internal cpHalo linker connecting the C-terminus of the N-terminal first effector sequence moiety to the N-terminus of the C-terminal first effector sequence moiety, The first partial effector sequence comprises or consists of: a sensor module array, to which said first partial effector array is connected, a second partial effector sequence consisting of a sequence selected from the sequences SEQ ID NO: 006 (PEP1) and SEQ ID NO: 007 (PEP2), to which said sensor module sequence is connected, Including, the first and second partial effector sequences together comprise a circularly permuted haloalkane dehalogenase and, when in close proximity to one another, are capable of effecting covalent attachment of a halogenalkane moiety; and said sensor module array comprising: a. a single sensor polypeptide capable of undergoing a conformational change from a first conformation to a second conformation in response to the presence or concentration of an analyte compound; When the first partial effector sequence is attached to the C-terminus of the sensor module sequence (the single sensor polypeptide) and the second partial effector sequence is attached to the N-terminus of the sensor module sequence (the single sensor polypeptide), in the first conformation, the first and second partial effector sequences are in close proximity (causing the first and second partial effector sequences to constitute a catalytically active entity); and in the second conformation, the first and second partial effector sequences are not adjacent (causing the first and second partial effector sequences to constitute a catalytically inactive entity); the single sensor polypeptide, and b. a sensor polypeptide pair comprising a first sensor polypeptide and a second sensor polypeptide, the first sensor polypeptide covalently linked to the first partial effector sequence via a peptide bond, and the second sensor polypeptide covalently linked to the second partial effector sequence; the first sensor polypeptide and the second sensor polypeptide are capable of specific intermolecular interaction (protein-protein binding); and the first and second sensor polypeptides are part of separate polypeptide chains. A sensor module array selected from The modular polypeptide.

2. The first partial effector sequence and the second partial effector sequence, when in close proximity to each other, exhibit a 10% or greater affinity for N-(10-(2-carboxy-5-((2-(2-((6-chlorohexyl)oxy)ethoxy)ethyl)carbamoyl)phenyl)-7-(dimethylamino)-9,9-dimethylanthracen-2(9H)-ylidene)-N-methylmethanaminium in a fluorescence polarization assay using N-(10-(2-carboxy-5-((2-(2-((6-chlorohexyl)oxy)ethoxy)ethyl)carbamoyl)phenyl)-7-(dimethylamino)-9,9-dimethylanthracen-2(9H)-ylidene)-N-methylmethanaminium as a substrate. ２ s －１ M －１ The modular polypeptide of claim 1, characterized by the activity of:

3. 2. The modular polypeptide of claim 1, wherein the first partial effector sequence and the second partial effector sequence have at least 0.5% of the activity of SEQ ID NO:001 when adjacent to each other.

4. The first partial effector sequence comprises: a. SEQ ID NO: 004, or b. a sequence at least 90% identical to SEQ ID NO: 004, or c. a sequence at least 90% identical to a construct consisting of SEQ ID NO:002 linked by a linker to SEQ ID NO:003; comprising or consisting of The modular polypeptide of any one of claims 1 to 3, wherein the first and second partial effector sequences together are characterized by at least 0.5% of the activity of SEQ ID NO:004 together with SEQ ID NO:007 (PEP2).

5. - the first partial effector sequence is connected to the sensor module sequence by a first inter-module linker sequence, and / or - said second partial effector sequence is connected to said sensor module sequence by a second inter-module linker sequence; A modular polypeptide according to any one of claims 1 to 4.

6. The modular polypeptide of any one of claims 1 to 5, wherein the sensor module sequence is a single sensor polypeptide consisting of an N-terminal first partial sensor sequence and a C-terminal second partial sensor sequence connected by a sensor linker sequence.

7. the first partial sensor sequence and the second partial sensor sequence are selected from a calmodulin binding peptide and a calmodulin polypeptide; the first partial sensor sequence is a calmodulin polypeptide and the second partial sensor sequence is a calmodulin binding peptide; said calmodulin polypeptide is or comprises SEQ ID NO: 009 (CaM) or a sequence that is at least 90% identical to SEQ ID NO: 009 (CaM) and has the same biological activity, and said calmodulin binding peptide is or comprises SEQ ID NO: 008 (M13); The sensor module array comprises: a. a first sensor peptide that is or comprises a calmodulin-binding peptide, the calmodulin-binding peptide being or comprising SEQ ID NO: 008 (M13); and b. a second sensor polypeptide that is or comprises a calmodulin polypeptide, said calmodulin polypeptide being or comprising SEQ ID NO: 009 (CaM) or a sequence that is at least 90% identical to SEQ ID NO: 009 (CaM) and has the same biological activity; The sensor polypeptide pair comprises the first sensor peptide is covalently linked to the first partial effector sequence via a peptide bond, and the second sensor polypeptide is covalently linked to the second partial effector sequence, and the first and second sensor polypeptides are part of separate polypeptide chains; the first partial effector sequence is connected to the C-terminus of the first sensor peptide by a first inter-modular linker sequence having 2 to 6 amino acids, and / or the second partial effector sequence is connected to the N-terminus of the second sensor polypeptide by a second inter-modular linker sequence having 2 to 6 amino acids; 7. The modular polypeptide of claim 6, wherein the first inter-modular linker sequence and the second inter-modular linker sequence are dipeptides or tripeptides, the amino acid components of which are each independently selected from G, S and T residues.

8. 8. The modular polypeptide of claim 7, characterized by a first polypeptide sequence consisting of or comprising SEQ ID NO:010 (SPLT1) or a sequence at least 90% identical to SEQ ID NO:010 (SPLT1), and a second polypeptide sequence consisting of or comprising SEQ ID NO:011 (SPLT2) or a sequence at least 90% identical to SEQ ID NO:011 (SPLT2), wherein the first and second polypeptide sequences together have at least 0.5% of the combined activity of SEQ ID NO:010 (SPLT1) and SEQ ID NO:011 (SPLT2).

9. The sensor module sequence is, from N-terminus to C-terminus: a. a calmodulin polypeptide, comprising SEQ ID NO:009 (CaM), or a sequence that is at least 90% identical to SEQ ID NO:009 (CaM) and has the same biological activity; b. a peptide linker, which is a polyproline type rigid helix, Pn proline polypeptide, where n is selected from an integer between 15 and 35, and optionally flanked by 1 to 4 amino acids; c. A calmodulin-binding peptide (second partial sensor sequence) comprising or consisting of SEQ ID NO: 008 (M13); The present invention is composed of a single sensor polypeptide comprising 9. The modular polypeptide of claim 7 or 8, comprising or consisting of a sequence selected from SEQ ID NO:013 (CONF1) and SEQ ID NO:014 (CONF2), or a sequence that is at least 90% identical to SEQ ID NO:014 (CONF2) or SEQ ID NO:013 (CONF1) having at least 0.5% of the activity of SEQ ID NO:013 (CONF1).

10. The sensor module array includes: a. an N-terminal portion of a glutamate binding protein, wherein the first sensor polypeptide is or comprises SEQ ID NO: 020 (GLT1), or a sequence that is at least 90% identical to SEQ ID NO: 020 (GLT1); and a C-terminal portion of a glutamate binding protein, and said second sensor polypeptide is or comprises SEQ ID NO:021 (GLT2), or a sequence that is at least 90% identical to SEQ ID NO:021 (GLT2) and has the same biological activity; wherein the combination of said first sensor polypeptide and said second sensor polypeptide has the same biological activity as the combination of SEQ ID NO: 020 (GLT1) and SEQ ID NO: 021 (GLT2); or b. A sequence that is at least 90% identical to a construct consisting of SEQ ID NO:020 (GLT1) linked to SEQ ID NO:021 (GLT2) by a polypeptide linker, said sensor module sequence being or comprising SEQ ID NO:022 (GLT3), or a sequence that is at least 90% identical to and has the same biological activity as SEQ ID NO:022 (GLT3); comprising or consisting of 5. The modular polypeptide of any one of claims 1 to 4, characterized by a first polypeptide sequence consisting of or comprising SEQ ID NO: 023 (GLTIND1), or a sequence at least 90% identical to SEQ ID NO: 023 (GLTIND1), and a second polypeptide sequence consisting of or comprising SEQ ID NO: 024 (GLTIND2), or a sequence at least 90% identical to SEQ ID NO: 024 (GLTIND2), wherein the first and second polypeptide sequences together have at least 0.5% of the activity of SEQ ID NO: 025 (GLTIND3).

11. The sensor module array comprises: a. a first sensor polypeptide that is or comprises an FKBP12 polypeptide, said FKBP12 polypeptide being or comprising SEQ ID NO: 015 (FKBP) or a sequence that is at least 90% identical to SEQ ID NO: 015 (FKBP) and has the same biological activity; and b. a second sensor polypeptide that is or comprises a FRB peptide, said FRB peptide being or comprising SEQ ID NO:016 (FRB) or a sequence that is at least 90% identical to SEQ ID NO:016 (FRB) and has the same biological activity; The sensor polypeptide pair comprises the first sensor polypeptide is covalently linked to the first partial effector sequence via a peptide bond, and the second sensor polypeptide is covalently linked to the second partial effector sequence, and the first and second sensor polypeptides are part of separate polypeptide chains; the first partial effector sequence is connected to the C-terminus of the first sensor polypeptide by a first inter-modular linker sequence having 2 to 9 amino acids, and / or the second partial effector sequence is connected to the N-terminus of the second sensor polypeptide by a second inter-modular linker sequence having 2 to 9 amino acids; the first inter-module linker sequence and the second inter-module linker sequence are tripeptides, the amino acid components of which are each independently selected from G, S and T residues; A modular polypeptide according to any one of claims 1 to 5.

12. The modular polypeptide of claim 11, characterized by a first polypeptide sequence consisting of or comprising SEQ ID NO: 017 (RAPIND1), or a sequence at least 90% identical to SEQ ID NO: 017 (RAPIND1), and a second polypeptide sequence selected from SEQ ID NO: 018 (RAPIND2) and SEQ ID NO: 019 (RAPIND3), or a sequence at least 90% identical to SEQ ID NO: 018 (RAPIND2), wherein the first and second polypeptide sequences together have at least 50% of the activity of the combination of SEQ ID NO: 017 (RAPIND1) and SEQ ID NO: 018 (RAPIND2).

13. One or more nucleic acids encoding a modular polypeptide according to any one of claims 1 to 12.

14. A combination of nucleic acids comprising: a. a first nucleic acid sequence encoding a first partial effector sequence, said encoded first partial effector sequence comprising, from N-terminus to C-terminus: i. SEQ ID NO:002, or a sequence at least 90% identical to SEQ ID NO:002; ii. a polypeptide linker sequence consisting of the sequence of SEQ ID NO: 005; iii. SEQ ID NO: 003, or a sequence at least 90% identical to SEQ ID NO: 003; the first nucleic acid comprising: b. a second nucleic acid encoding a second partial effector sequence characterized by SEQ ID NO: 006 (PEP1) or 007 (PEP2); Including, A combination of said nucleic acids, wherein said first and said second partial effector sequences together comprise a circularly permuted haloalkane dehalogenase and, when in close proximity to one another, are capable of effecting covalent attachment of a haloalkane moiety.

15. a. The nucleic acid of claim 13, or b. a first nucleic acid according to claim 14, and a second nucleic acid according to claim 14; wherein each nucleic acid is under the control of a promoter sequence.

16. A cell comprising the nucleic acid expression system of claim 15, wherein the promoter is operable in the cell.

17. 16. A non-human transgenic animal or plant comprising a nucleic acid according to claim 13, a combination of nucleic acids according to claim 14, or a nucleic acid expression system according to claim 15.

18. A kit comprising a nucleic acid or a nucleic acid expression system according to any one of claims 13 to 15 and a Halotag 7 substrate.

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