New method for transducing protein-protein interactions

Abstract

The present invention relates to a cell comprising a first nucleic acid sequence encoding a first polypeptide fused to the N-terminus of a first variant of a histidine kinase comprising a DHp domain and a CA domain, wherein said first variant does not comprise a transmembrane domain, a second nucleic acid sequence encoding a second polypeptide fused to the N-terminus of a second variant of said histidine kinase comprising a DHp domain and a CA domain, wherein said second variant does not comprise a transmembrane domain, and a third nucleic acid sequence encoding a response regulator protein that can be specifically phosphorylated by said DHp domain of said first or said second variant. The present invention further relates to the use of a cell of the present invention.

Description

[0001] This application claims the benefit of priority to European patent application EP18200357.4, filed on 15 October 2018, which is incorporated herein by reference.

[0002] This invention relates to methods and means for evaluating or responding to protein-protein interactions and transducing such interactions to the expression of a target gene. Background Technology

[0003] Molecular interactions, such as protein / protein interactions, are involved in almost every cellular process in living cells. Characterizing protein / protein interactions is a crucial step in better understanding and controlling biological systems. Transducing protein / protein interactions to detectable signals or target gene expression is important for new drug discovery and the development of novel cells for use as cell-based biosensors or cell therapies.

[0004] Different cellular pathways are modulated by the presence / absence of compounds that promote or inhibit protein / protein interactions. Identifying molecules that regulate protein / protein interactions is one of the main tools used in drug discovery and development. Furthermore, transducing protein / protein interactions into novel responses is a primary tool in cell engineering for therapeutic purposes.

[0005] An example of compound-regulated protein interactions is the interaction between G protein-coupled receptors (GPCRs) and downstream pathway proteins, such as β-repressor proteins. GPCRs are membrane receptors in mammalian cells that detect a variety of ligands (endogenous hormones, growth factors, and natural or synthetic small molecules). Following interaction between a GPCR and its ligand, various cascade reactions are induced in the cell, regulating cellular activity. Due to the central function of GPCRs in the cell, many drugs act on GPCRs as their targets. Various assays have been developed to identify and characterize GPCR agonists and antagonists. Some of these assays transduce the interaction of a GPCR with one of its protein couplers into the expression of a reporter gene.

[0006] Furthermore, protein / protein interactions have been used to design chimeric sensors that can sense different signals and transduce them to specific responses. This ability to redirect information from a defined input to a specific output can be used in many applications, such as generating cell-based biosensors to create new in vitro diagnostic tools or new cell therapies with improved safety and efficacy. Despite significant developments in the biosensor field, many merely generate detectable signals that require manual readout and interpretation. Progress in biosensors that transduce signals into downstream biological activities, such as gene expression, has been considerably less. To date, described artificial signal transduction systems primarily utilize the cleavage of fusion proteins to release transcription activators, which then regulate the expression of target genes.

[0007] Following ligand-triggered autophosphorylation of the histidine kinase (HK) receptor protein at a histidine residue, the phosphoryl group is then transferred to an aspartic acid residue in the response regulator (RR) protein, initiating the two-component system (TCS) signaling cascade. With some exceptions, the HK sensor forms a homodimer in the cell membrane. The structural basis for HK autophosphorylation is the existence of two distinct HK dimer conformations. In the unstimulated state, the conformation is such that the catalytic ATP-binding (CA) domain is located away from the histidine residue in the dimer and the histidine-containing phosphotransferase (DHp) domain is located away from the histidine residue in the dimer. Figure 1 a) Therefore, autophosphorylation does not occur. Upon ligand binding, the CA domain and bound ATP enter the vicinity of the histidine residue of DHp, resulting in phosphorylation. Subsequently, the homologous response regulator (RR) binds to the phosphorylated DHp domain, and phosphate is transferred from the histidine residue in the acceptor domain of the RR to an aspartate residue. The phosphorylated RR then binds to its target promoter and regulates gene expression. Furthermore, when the phosphorylated RR binds to the unphosphorylated DHp domain, the latter catalyzes the dephosphorylation of the aspartate residue, thus actively shutting down signal transduction. Therefore, HK is a bifunctional enzyme with both kinase and phosphatase activities, and the balance between the two determines the signal strength and kinetics.

[0008] Due to the importance of GPCR signaling in human diseases, various assays have been developed to detect and identify molecules that interact with GPCRs. Among the different methods developed, some utilize the fact that β-repressor proteins interact with ligand-activated GPCRs. Bioluminescent resonance energy transfer (BRET) analysis, TANGO analysis (Invitrogen)... Figure 5The ChaCha system and the ChaCha system are examples of these experiments. The first system transduces protein / protein interactions into detectable fluorescent signals. The latter two experiments transduce protein / protein interactions into the expression of target genes (such as reporter genes).

[0009] TANGO analysis was performed by fusing a proteolytically cleavable artificial transcription factor (GAL4-VP16) to the intracellular domain of a GPCR, and by fusing a TEV protease with a β-repressor protein. Ligand activation of the GPCR induced the recruitment of the β-repressor protein to the GPCR, bringing the TEV protease into close proximity to the cleavable linker on the GPCR and allowing the release of GAL4-VP16. The artificial transcription factor then induced the expression of a reporter gene (β-lactamase) driven by a chimeric promoter targeted by GAL4-VP16.

[0010] The ChaCha system has recently been developed as a derivative of TANGO assays. In this system, dCas9 (which cannot cleave DNA but can still bind to it), linked to a triplet of transcriptional activators consisting of VP64, p65 activation domains, and Rta (dCas9-VRP), fuses with a β-repressor protein, while the intracellular domain of the GPCR fuses with a TEV protease. This system also requires the expression of guide RNA (gRNA), which allows dCas9 to be recruited to the promoter, driving the expression of the target gene. The interaction between the GPCR and the β-repressor protein-dCas9-VRP fusion releases dCas9-VRP. dCas9-VRP regulates the expression of the target gene targeted by the co-expressed gRNA in the cell. The promoter can be endogenous or chimeric.

[0011] Different methods have been developed for detecting general protein-protein interactions, such as those in the cytoplasm. One of the most popular methods is the yeast two-hybrid approach. In this method, two potentially interacting proteins, often referred to as bait and prey, are fused to a split subunit of a protein with a specific detectable biological activity. Each individual split subunit does not exhibit the biological activity in question. The interaction between bait and prey allows for the appropriate reconstruction of the domains fused separately to the bait and prey. Different reporter systems have been developed based on the localization of the bait and prey.

[0012] - In order to detect nuclear localization, the splitting protein reactivates the transcriptional activation of the reporter gene;

[0013] - For protein-protein interactions that occur in the cytoplasm or on the membrane, the reporter system is based on yeast growth by activating Ras signaling, uracil auxotrophs, or antibiotic resistance.

[0014] One drawback of the yeast two-hybrid method is that the interactions are quantified within yeast cells, and they may not accurately reproduce the interactions in the natural mammalian cellular environment.

[0015] Therefore, the object of the present invention is to provide means and methods for responding to and / or evaluating protein-protein interactions. Summary of the Invention

[0016] This objective is achieved by the cells and methods specified in the independent claims. Advantageous embodiments are set forth in the dependent claims and the following description.

[0017] A first aspect of the invention relates to a recombinant cell. The cell facilitates the analysis of interactions between a pair of polypeptides or proteins. These interacting partners are hereinafter referred to as a "first polypeptide" and a "second polypeptide." Each of these polypeptides is encoded by a nucleic acid sequence, and each of these polypeptides is part of a fusion protein containing a polypeptide portion for analyzing its interactions with other polypeptides, and a fragment of a histidine kinase variant that retains DHp and CA activity.

[0018] The cells of the present invention include

[0019] - The first nucleic acid sequence encodes a first polypeptide fused to the N-terminus of a first variant of histidine kinase (EC2.7.13.3). The histidine kinase comprises a DHp (dimerizing and histidine-containing phosphotransferase) domain and a CA (catalytic ATP-binding) domain. The cell further includes...

[0020] - A second nucleic acid sequence encoding a second polypeptide fused to the N-terminus of a second variant of a histidine kinase, the histidine kinase comprising a DHp domain and a CA domain, and

[0021] - A third nucleic acid sequence that encodes a response regulatory protein that can be specifically phosphorylated by the DHp domain of the first or second variant of the histidine kinase.

[0022] In other words, this aspect of the invention relates to a cell comprising

[0023] - A first nucleic acid sequence, wherein the first nucleic acid sequence encodes...

[0024] In the N-to-C direction, the interaction between the first polypeptide and the second polypeptide is analyzed. The first polypeptide is fused to the N-terminus of a first variant of a histidine kinase, wherein the histidine kinase comprises a DHp domain and a CA domain, and both the DHp domain and the CA domain are retained in the first variant.

[0025] - A second nucleic acid sequence encoding a second polypeptide fused to the N-terminus of the second variant of the histidine kinase, the histidine kinase comprising a DHp domain and a CA domain, wherein the DHp domain and CA domain are also retained in the second variant.

[0026] - A third nucleic acid sequence that encodes a response regulatory protein that can be specifically phosphorylated by the DHp domain of the first or second variant.

[0027] In a particular embodiment, the first and second polypeptides do not contain any portion of the aforementioned histidine kinase, and in particular, do not contain a transmembrane domain, a sensor domain, or a transmitter domain.

[0028] In certain embodiments of the cells of the present invention, the first variant and / or the second variant do not contain the transmembrane domain of histidine kinase.

[0029] In some embodiments of the cells of the present invention

[0030] - The first variant does not contain a functional histidine kinase carrier domain and / or a functional sensor domain, and / or;

[0031] - The second variant does not contain a functional histidine kinase delivery domain and / or a functional sensor domain.

[0032] In a particular implementation, the naturally occurring sensor and transducer domains of histidine kinase are replaced by two target proteins whose interactions should be evaluated. If specific interactions exist between these proteins, their binding promotes dimerization of the truncated histidine kinase variant, thereby achieving spatial proximity between the ATP-containing CA domain and the DHp domain. This results in phosphorylation of the DHp domain, which is then capable of phosphorylating the cognate, response regulatory domain, and particularly the receptor domain of the response regulatory protein.

[0033] Alternatively, the two target proteins can form an artificial signal transduction pathway, where the binding of the two target proteins is triggered by a stimulus, such as a ligand that can be specifically recognized by one or both target proteins. Ligand recognition can be associated with a desired response mediated by the activity of response-regulating proteins. This response can be a microRNA affecting cellular processes or the expression of proteins (e.g., cytokines or antibodies). In a particular embodiment, the cell can be used for medical applications, where a beneficial or therapeutic response can be specifically triggered by, for example, a disease-related compound (such as a disease-related antigen).

[0034] Alternatively, the effects of the compounds on known interacting proteins can be assessed using the cells of the present invention, wherein the effects of the compounds can be determined by the activity of response regulatory proteins.

[0035] In particular, response regulatory proteins contain effector functions that can be identified for assessing the interaction between a first peptide and a second peptide, or for evoking a desired response to the aforementioned stimuli. Non-limiting examples of such effector functions include binding to DNA, RNA, or enzymes (e.g., enzymes that catalyze the formation of cAMP).

[0036] In a particular implementation, the effector function includes specific binding to the promoter sequence and inducing the expression of the target gene.

[0037] In certain embodiments of the cell of the present invention, the response regulatory protein comprises a receptor domain fused to an effector domain, wherein the receptor domain is phosphorylated by the DHp domain of a first or second histidine kinase variant, and the effector domain is modulated by the phosphorylated receptor domain, specifically activated or inhibited. In particular, the activity of the effector domain varies depending on the phosphorylation state of the receptor domain; therefore, the activity of the effector domain can be increased, activated, decreased, or inhibited by phosphorylation of the receptor domain.

[0038] In some embodiments of the cells of the present invention

[0039] - The effector domain is the transcriptional activation domain, and

[0040] - The cell contains a fourth nucleic acid sequence, which is contained in a target gene controlled by an inducible promoter that can be recognized by a transcriptional activation domain.

[0041] The expression of the target gene is induced after the transcriptional activation domain is activated.

[0042] In some embodiments of the cells of the present invention, the target gene encodes a target protein, particularly a fluorescent or luminescent protein, or a target RNA.

[0043] The target protein can be a fluorescent or luminescent protein, thereby allowing the successful interaction between the first and second peptides to be determined or observed by the fluorescence or luminescence of the target protein.

[0044] Alternatively, the target protein or target RNA may be or trigger a desired response to the aforementioned stimuli, such as a desired therapeutic response (cytokine, antibody, reactive oxygen species production, etc.).

[0045] In some embodiments of the cells of the present invention

[0046] - The first and second variants are variants of EnvZ kinase (UniProt No P0AEJ4), and the response regulatory protein includes or is an OmpR response regulatory (RR) protein (Uniprot No P0AA16), or

[0047] - The first and second variants are variants of NarX kinase (UniProt No P0AFA2), the receptor domain contains or is a NarL response regulation (RR) protein (Uniprot No. P0AF28), and the effector domain is or contains a VP16 transcriptional activation domain (Vp48, SEQ ID 7).

[0048] As mentioned earlier, the effector architecture domain can be a portion of the NarL fused with VP16.

[0049] In some embodiments of the cell of the present invention, the transcriptional activation domain is composed of or contains amino acids characterized by SEQ ID NO 7.

[0050] In certain embodiments of the cell of the present invention, the first or second variant is or comprises a selection from EnvZ. 180-450 (SEQ ID 8), EnvZ 223-450 (SEQ ID 9), NarX 176-598 (SEQ ID 10) and NarX 379-598 A variant of (SEQ ID 11), or a functionally equivalent polypeptide to any one of SEQ ID 8-11 having at least 70%, 80%, 85%, 90%, 95%, 98%, or 99% sequence identity.

[0051] In some embodiments of the cells of the present invention, the inducible promoter is selected from the OmpR promoter (SEQ ID 1) and the NarL-RE promoter (SEQ ID 2).

[0052] In some embodiments of the cell of the present invention, the first nucleic acid sequence and / or the second nucleic acid sequence and / or the third nucleic acid sequence are optimized for the cell’s codon usage.

[0053] In some embodiments of the cells of the present invention, the first nucleic acid sequence and / or the second nucleic acid sequence and / or the third nucleic acid sequence are under the transcriptional control of a constitutive promoter.

[0054] In some embodiments of the cells of the present invention, the constitutive promoter is selected from CMV (SEQ ID 3), EF1α (SEQ ID 4) and EF1α-V1 (SEQ ID 5).

[0055] In some embodiments of the cells of the present invention, the first variant and the second variant are identical.

[0056] In some embodiments of the cells of the present invention, histidine kinase belongs to the transphosphorylation family.

[0057] In some embodiments of the cells of the present invention

[0058] - The first variant contains a DHp domain that does not contain histidine residues accessible to the CA domain of the first or second variant of the histidine kinase, and / or

[0059] - The second variant contains a CA domain that cannot bind to ATP.

[0060] In some embodiments of the cells of the present invention

[0061] - The first variant is or contains the variant NarX 379-598 (H399Q)(SEQ ID 12) or a functionally equivalent polypeptide having at least 70%, 80%, 85%, 90%, 95%, 98%, or 99% sequence identity with (SEQ ID 12), and / or - a second variant is or contains variant NarX. 379-598 (N509A)(SEQ ID 13) or a functionally equivalent polypeptide having at least 70%, 80%, 85%, 90%, 95%, 98% or 99% sequence identity with (SEQ ID 13).

[0062] In some embodiments of the cells of the present invention, the specific binding of the first polypeptide and the second polypeptide may be triggered by a ligand specifically recognized by the first and / or second polypeptide.

[0063] In some embodiments of the cells of the present invention, the first polypeptide is a receptor or contains a receptor, and the second polypeptide is a binding partner of the receptor or contains a binding partner of the receptor, wherein the binding of the receptor and the binding partner can be triggered by a receptor-recognizable ligand.

[0064] In some embodiments of the cells of the present invention, the receptor is a transmembrane receptor, and the binding partner is a cytoplasmic protein. In this case, the ligand that can be recognized by the receptor and the cytoplasmic protein that serves as the binding partner are specifically separated by the membrane.

[0065] In some embodiments of the cells of the present invention

[0066] - The first polypeptide consists of or contains a G protein-coupled receptor, and the second polypeptide consists of or contains a cytoplasmic ligand of the G protein-coupled receptor, particularly β-repressor protein, or

[0067] - The first polypeptide is composed of or contains one of the T cell receptors or their components, and the second polypeptide is or contains a cytoplasmic ligand of the T cell receptors or their components, particularly ZAP-70 (UniProt NoP43403).

[0068] In some embodiments of the cell of the present invention, the cell is a mammalian cell, particularly a human cell.

[0069] Another aspect of the present invention relates to a method for evaluating protein-protein interactions. The method includes:

[0070] -Providing a cell according to the invention, said cell comprising:

[0071] • A first nucleic acid sequence encoding a first polypeptide fused to the N-terminus of a first variant of a histidine kinase (EC2.7.13.3), the histidine kinase comprising a DHp domain and a CA domain.

[0072] • A second nucleic acid sequence encoding a second polypeptide fused to the N-terminus of a second variant of histidine kinase, the histidine kinase comprising a DHp domain and a CA domain, and

[0073] • A third nucleic acid sequence encoding a response regulatory protein that can be specifically phosphorylated by the DHp domain of the first or second variant; and

[0074] - Determine the activity of the response regulation (RR) protein.

[0075] Upon specific binding of the first and second peptides, the first and second variants dimerize, causing the CA domain of the first or second variant to phosphorylate the DHp domain of the first or second variant, and thereby modulate, in particular activate or inhibit, the activity of response regulatory proteins through phosphorylation of the DHp domain of the first or second variant.

[0076] Another aspect of the present invention relates to a method for determining the effect of a compound on protein-protein interactions. The method includes:

[0077] -Provide a cell according to the invention, said cell comprising,

[0078] • The first nucleic acid sequence encodes a first polypeptide fused to the N-terminus of a first variant of histidine kinase, the histidine kinase comprising a DHp domain and a CA domain.

[0079] • A second nucleic acid sequence encoding a second polypeptide fused to the N-terminus of a second variant of histidine kinase, the histidine kinase comprising a DHp domain and a CA domain, and

[0080] • A third nucleic acid sequence encoding a response regulatory protein that can be specifically phosphorylated by the DHp domain of the first or second variant, and

[0081] -Contacting cells with compounds, and

[0082] - Determine the activity of the response regulatory protein.

[0083] in

[0084] Upon specific binding of the first and second peptides, the first and second variants dimerize, causing the CA domain of the first or second variant to phosphorylate the DHp domain of the first or second variant, and thereby regulating, in particular activating or inhibiting, the activity of the response regulatory protein by phosphorylation of the DHp domain of the first and / or second variants.

[0085] The effect of compounds on the specific binding of the first and second polypeptides is determined by the activity of response regulatory proteins.

[0086] Advantageously, the above method can be used as a screening assay to evaluate the effect of any compound on any target protein-protein interaction.

[0087] Another aspect of the present invention provides a method for eliciting a desired response in response to a stimulus. The method according to this aspect of the invention includes the following steps:

[0088] -Provide a cell according to the invention, said cell comprising,

[0089] • The first nucleic acid sequence encodes a first polypeptide fused to the N-terminus of a first variant of histidine kinase, the histidine kinase comprising a DHp domain and a CA domain.

[0090] • A second nucleic acid sequence encoding a second polypeptide fused to the N-terminus of a second variant of the histidine kinase, the histidine kinase comprising a DHp domain and a CA domain.

[0091] The specific binding of the first and second polypeptides is triggered by a stimulus, and

[0092] • A third nucleic acid sequence encoding a response regulatory protein that can be specifically phosphorylated by the DHp domain of the first or second variant.

[0093] Upon specific binding of the first and second peptides, the first and second variants dimerize, causing the CA domain of the first or second variant to phosphorylate the DHp domain of the first or second variant. This phosphorylation of the DHp domain of the first and / or second variant regulates, in particular, the activity of response regulatory proteins, and...

[0094] - Exposing cells to stimuli in which the desired response is mediated by the activity of response regulatory proteins or the activity of response regulatory proteins.

[0095] Preferably, the first or second polypeptide is a receptor that recognizes a stimulus, such as a T-cell receptor that recognizes a disease-associated antigen, or a G-coupled receptor, wherein the other polypeptide may be a ligand of the receptor, such as β-inhibitory protein or ZAP-70, respectively.

[0096] The term "specific binding of the first and second polypeptides" specifically refers to Kd less than 10. -5 M, 10 -6 M, 10 -7 M, 10 -8 M or 10 -9 The combination of M.

[0097] In some embodiments of the method of the present invention, the response regulatory protein comprises a receptor domain fused to an effector domain, wherein the receptor domain may be phosphorylated by a first or second variant of the DHp domain, and the effector domain may be activated by the phosphorylated receptor domain.

[0098] In some embodiments of the method of the present invention

[0099] - The response regulatory protein includes a receptor domain fused to an effector domain, wherein the receptor domain may be phosphorylated by the DHp domain of the first or second variant, and the effector domain may be activated by the phosphorylated receptor domain.

[0100] - The effector domain is a transcriptional activation domain.

[0101] The cell also contains a fourth nucleic acid sequence encoding a target gene, which is controlled by an inducible promoter recognized by the transcriptional activation domain.

[0102] -The expression of the target gene is induced after the transcriptional activation domain is activated.

[0103] In some embodiments of the method of the present invention, the presence of the target gene expression product is determined as the activity of the response regulatory protein.

[0104] In some embodiments of the method of the present invention, the expression product of the target gene has optical properties, such as containing a luminescent portion, as in the case of green fluorescent protein (GFP).

[0105] In some embodiments of the method of the present invention, the expression product of the target gene is Cerulean.

[0106] In some embodiments of the method of the present invention, the expression product of the target gene is or mediates a desired response. For example, the expression product may be a cytokine designed to elicit an immune response in the cell. The expression product may also be a component of a signaling cascade that elicits a desired response further downstream in the cascade. The expression product may also be RNA capable of triggering the desired response, such as microRNA or guide RNA for self-regulating endogenous genes.

[0107] Furthermore, this invention provides a vector particularly suitable for transfecting or transducing mammalian cells, especially human cells. The vector comprises:

[0108] - The first nucleic acid sequence contained within the cells of this invention

[0109] - A second nucleic acid sequence contained within the cells of this invention,

[0110] - The third nucleic acid sequence contained within the cells of this invention, and

[0111] -Optionally included in the cells of the present invention a fourth nucleic acid sequence.

[0112] According to another aspect of the invention, a fusion protein is provided. The variant comprises a polypeptide fused to a variant of a histidine kinase (EC2.7.13.3), the histidine kinase comprising a DHp (dimerization and histidine-containing phosphate transfer) domain and a CA (catalytic ATP-binding) domain.

[0113] Specifically, the polypeptide does not contain any portion of the aforementioned histidine kinase, and particularly does not contain a transmembrane domain, a sensor domain, or a transtransmitter domain.

[0114] In some embodiments of the fusion protein of the present invention, the variants do not contain the transmembrane domain of histidine kinase.

[0115] In some embodiments of the fusion protein of the present invention, the variants do not contain a functional histidine kinase delivery domain and / or a functional sensor domain.

[0116] In some embodiments of the fusion protein of the present invention, the variant is a variant of EnvZ kinase (UniProt NoP0AEJ4) or a variant of NarX kinase (UniProt No P0AFA2).

[0117] In certain embodiments of the fusion protein of the present invention, variants are or contain selections from EnvZ. 180-450 (SEQ ID8), EnvZ 223-450 (SEQ ID 9), NarX 176-598 (SEQ ID 10) and NarX 379-598A variant of (SEQ ID 11), or a functionally equivalent polypeptide to any one of SEQ ID 8-11 having at least 70%, 80%, 85%, 90%, 95%, 98%, or 99% sequence identity.

[0118] In some embodiments of the fusion protein of the present invention, histidine kinase belongs to the transphosphorylation family.

[0119] In some embodiments of the fusion protein of the present invention, variants include a DHp domain that does not contain histidine residues accessible to the CA domain of a variant of histidine kinase or another variant, or contains a CA domain that cannot bind ATP.

[0120] In certain embodiments of the fusion protein of the present invention, the variant is or includes the variant NarX. 379-598 (H399Q)(SEQ ID 12), variant NarX 379-598 (N509A)(SEQ ID 13) or a functionally equivalent polypeptide having at least 70%, 80%, 85%, 90%, 95%, 98% or 99% sequence identity with SEQ ID 12 or 13.

[0121] In some embodiments of the fusion protein of the present invention, the polypeptide consists of or contains a G protein-coupled receptor, or consists of or contains a cytoplasmic ligand of a G protein-coupled receptor, particularly a β-repressor protein.

[0122] In some embodiments of the fusion protein of the present invention, the polypeptide is composed of or contains one of the T cell receptors or components thereof, or the polypeptide contains a cytoplasmic ligand of the T cell receptors or components thereof, particularly ZAP-70 (UniProt No P43403).

[0123] The present invention is further illustrated by the following embodiments and accompanying drawings, from which further embodiments and advantages can be derived. These embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Attached Figure Description

[0124] Figure 1A schematic diagram of the two-component signaling pathways, both native and transplanted, is shown. (A) The native pathway consists of a receptor histidine kinase (HK) protein, which, in the presence of its signal, autophosphorylates and phosphorylates its homologous response regulator (RR) at the level of an asparagine present in the receptor domain. The phosphorylated RR binds to a specific response element present in the promoter regulated by the RR. (B) The TCS transplanted into the mammalian host is expressed by a gene with a human-optimized codon sequence. The histidine kinase transplanted into mammalian cells is always active and autophosphorylates and phosphorylates the RR. The transplanted RR is enhanced by the VP48 transactivation domain. The phosphorylated RR binds to the RE present in the engineered promoter, which drives the expression of the target gene, in this case, the fluorescent reporter cerulean. DNB, DNA-binding domain. VP48, VP48 transactivation domain; Pmin, minimal mammalian promoter.

[0125] Figure 2 Showing cis-to-trans autophosphorylation of HK. (A) Schematic diagram of cis-autophosphorylation (upper lane) and trans-autophosphorylation (lower lane) in mammalian cells expressing WT HK (first lane), DHp mutant (second lane), CA mutant (third lane), or DHp mutant and CA mutant (fourth lane). (B) Quantitative data of mammalian cells expressing the reporter gene, either solely or in combination with wild-type HK, DHp mutants (EnvZ H243V, NarX H399Q, and DcuS H350L), CA mutants (EnvZ N347A, NarX N509A, and DcuS N445A), or DHp mutant and CA mutant. Bar charts show Cerulean levels as mean ± SD of independent biological triplicates, normalized to expression in transfected controls (norm.u.).

[0126] Figure 3 Demonstrating the activity of truncated HK. (A) Schematic diagram of phosphate transfer between the EnvZ / OmpR element (upper lane) and the NarX / NarL element (lower lane) in mammalian cells expressing WT HK (first column), a truncated sensor domain mutant (second column), or a truncated sensor and transmitter domain mutant (third column). (B) Quantitative data from mammalian cells expressing a reporter gene, wherein the mammalian cells express only the reporter gene or, in combination with wild-type HK, a truncated sensor domain mutant (EnvZ / OmpR / NarL / NarL), or a truncated sensor domain mutant (EnvZ / OmpR / NarL ... 180-450 and NarX 176-598 ), or truncated mutants of sensor and transmitter structural domains (EnvZ) 223-450 and NarX 379-598Combined expression. The bar chart shows Cerulean levels as mean ± SD of independent biological triplicates, normalized to expression in the transfected control (norm.u.).

[0127] Figure 4 The design of the protein / protein interaction assay is shown. (A) Schematic diagram of PPI analysis used to monitor the interaction between two proteins, P1 and P2. The interaction between P1 and P2, spontaneously or induced by the compound, allows the transphosphorylated family HK short cytoplasmic domain mutated at the CA domain level fused with P1 to dimerize with the transphosphorylated family HK short cytoplasmic domain mutated at the DHp domain level fused with P2. Dimerization triggers phosphorylation of RR, which binds its RE and induces reporter gene expression. (B) Quantitative data of mammalian cells expressing HK mutants fused to the SZ1 or SZ2 domains. (C) Quantitative data of mammalian cells expressing HK mutants fused to the FKBP or FRB domains in the absence of (white bars) or in the presence of A / C heterodimers (black bars) that induce dimerization of the FKBP and FRB domains. The bar chart shows Cerulean levels as independent biological triplicate mean ± SD, normalized to expression in transfected controls (norm.u.).

[0128] Figure 5 The design of a protein / protein interaction (PPI) assay for GPCRs is shown. (A) Schematic diagram of a TANGO assay used to monitor the interaction between GPCRs and β-repressor proteins. The agonist-induced interaction between GPCRs and β-repressor proteins allows the β-repressor-TEV protease fusion complex to localize at the GPCR-tTA level and trigger the release of transcription factor tTA. (B) Schematic diagram of a PPI assay monitoring the interaction between GPCRs and β-repressor proteins. The agonist-induced interaction between GPCRs and β-repressor proteins allows the short cytoplasmic domain of the transphosphorylated family HK, mutated at the CA domain level fused with the GPCR, to dimerize at the DHp domain level fused with the β-repressor protein. Dimerization triggers phosphorylation of RR, which binds to its RE and induces reporter gene expression. (C) Quantitative data from mammalian cells expressing TANGO. (D) Quantitative data of mammalian cells expressing HK mutants fused to GPCRs or β-repressor proteins in absence (white bars) or in presence of procaterol (black bars). Bar charts show Cerulean levels as mean ± SD of independent biological triplicates, normalized to expression in transfected controls (norm.u.).

[0129] Figure 6 This illustrates the restoration of bicomponent signal transduction through forced dimerization of protein moieties fused to the C- or N-terminus of NarX. The figure shows signal levels in mammalian cells expressing the response regulator NarL alone and the NarL-regulated mCerulean fluorescent protein reporter, or in different combinations of SynZip1 and SynZip2 fused to the C- or N-terminus of NarX. The bar charts show Cerulean levels as independent biological triplicate mean ± SD, normalized to expression in transfected controls (norm.u.).

[0130] Figure 7 Comparison of CMV and EF1α promoters. (a) iRFP fluorescence in HEK cells transfected with plasmids expressing iRFP from the CMV promoter (white bar) or from the EF1α promoter (black bar). The figure shows reporter gene expression in DMEM without any ligands, or in the presence of 1 μM isopropylquinolone or 2 μM epinephrine. The bars show iRFP levels as independent biological triplicate mean ± SD, normalized to the frequency of transfection with the marker Citrine-positive cells (rel.u.). (b) NarX / NarL activity expressed by the CMV or EF1α promoters. The figure shows each transfection with a NarL-regulated mCerulean fluorescent protein reporter and plasmids expressing NarL and NarX from the CMV, EF1α, or EF1α-V1 promoters. The bars show Cerulean levels as independent biological triplicate mean ± SD, normalized to expression in transfected controls (norm.u.).

[0131] Figure 8 The restoration of bicomponent signal transduction is shown through forced dimerization via fusion with wild-type NarX or various NarX mutants. The figure illustrates signal levels in mammalian cells expressing the response regulator NarL alone and the NarL-regulated mCerulean fluorescent reporter, or with different combinations of SynZip1 and SynZip2 fused to wild-type NarX or various NarX mutants. The bar charts show Cerulean levels as independent biological triplicate mean ± SD, normalized to expression in transfected controls (norm.u.).

[0132] Figure 9The restoration of bicomponent signal transduction by forced dimerization is shown. As illustrated, signal levels in mammalian cells expressing the response regulator NarL alone and the NarL-regulated mCerulean fluorescent reporter, or only with a different variant of the NarX mutant fused to SynZip1 or SynZip2, are shown. The bar charts show Cerulean levels as independent biological triplicate mean ± SD, normalized to expression in transfected controls (norm.u.).

[0133] Figure 10 The transduction of gene expression by cytoplasmic ligand concentration is shown. (a) Signal levels in mammalian cells expressing the response regulator NarL alone and the NarL-regulated mCerulean fluorescent reporter, or only with a different variant of the NarX mutant fused to FKBP and FRB, as shown in the figure. For each pair of bars, the left bar (white) represents reporter expression without A / C ligands, and the right bar (black) represents reporter expression with ligands (100 nM). The bar chart shows Cerulean levels normalized to the expression of the transfected control (norm.u.). (b) Representative micrographs of the same transfection, as shown in the figure. Figure 3 As shown in b, the transfection control channel (red) is included. In all figures, the top and bottom rows of the image show the expression of mCherry transfection reporter (red false color) and the mCreulean protein output induced by the pathway with or without ligand in the same transfection (cyan false color), respectively. The bar charts show Cerulean levels as independent biological triplicate mean ± SD, normalized to the expression of the transfection control (norm.u.).

[0134] Figure 11 The rewiring of reporter gene expression by GPCR activity is shown. (a) Signal levels in mammalian cells expressing the response regulator NarL and the NarL-regulated mCerulean fluorescent reporter, as well as different combinations of the protein domains and their fusions shown. (b) Signal levels in mammalian cells expressing the CMV or EF1α-V1 promoter components as determined by TANGO. For each pair of bars in Figures a and b, the left bar (white) represents the absence of isopropylquinolone reporter expression, and the right bar (black) represents the presence of isopropylquinolone reporter expression (2 μM). The bar charts show Cerulean levels as the mean ± SD of independent biological triplicates, normalized to the expression of the transfected control (norm.u.). (c) Representative micrographs of transfections of the same selection, as shown in the image. Figure 4As shown, the expression of the mCherry transfection control is also displayed here. In all figures, the top and bottom rows of the image show the expression of the mCherry transfection reporter (red false color) and the output of the mCreulean protein induced by the same transfection pathway with or without ligand (cyan false color), respectively.

[0135] Detailed description of certain embodiments of the present invention

[0136] This invention provides a novel method for transducing protein / protein interactions into gene expression in mammalian cells using components from a two-component system present in bacteria. This invention can be used to develop new screening assays, generally for protein-protein interactions, particularly for GPCR signal regulation. Compared to existing technologies (e.g., the TANGO system from ThermoFischer), the system described herein is characterized by an excellent dynamic range and, due to its virtually unlimited supply of building blocks, has enormous potential for high-throughput multiplexing.

[0137] This invention provides a foundation for cell-based biosensors and engineered synthetic signal transduction modules in therapeutic cells, possessing properties such as low background levels, high dynamic range, and reversibility. Because the method can be multiplexed, it will allow the creation of complex logic-based circuits that can provide novel capabilities to modified cells.

[0138] Specifically, the present invention includes three distinct features:

[0139] -HK (histidine kinase) component (divided into two different mutants)

[0140] -RR components

[0141] - This gene construct contains a chimeric promoter that allows the expression of the target gene.

[0142] The HK domain fused to the two interacting proteins is mutated to increase dynamic range. Wild-type domains can also be used, but this has so far resulted in a lower dynamic range. However, the use of wild-type domains can offer some advantages when using this system to detect homodimerization. Additionally, in the case of homodimerization of the target protein, HKs belonging to the cis family can be used. An advantage of this method is the reduction in the number of genetic constructs. However, in all cases, a key feature of the invention is the fusion of the target protein with a reduced-size domain of the HK that does not dimerize on its own unless forced dimerization is achieved by means of the fusion component, and therefore does not transduce transcriptional activity itself.

[0143] The RR (response regulator) used in this experiment is fused with VP48 as a transcriptional activation domain that functions in mammalian cells. Other transcriptional activation domains can be fused with RRs, such as the p65(RelA) domain or Rta. In this embodiment, the inventors used an RR that directly binds to DNA, but other types of RRs can be used depending on the desired readout. For example, some RRs bind to RNA, while others are enzymes that catalyze the production of compounds that supply the secondary signal transduction chain, such as cyclic di-GMP.

[0144] The gene construct expressing the target gene (GOI) comprises two elements. The first part is a chimeric promoter. The inventors use a minimal promoter linked to an upstream sequence having numerous RR binding sites. The distance between the minimal promoter and the number of REs can be modified to upregulate or downregulate GOI expression.

[0145] The GOI used in the experiment is the fluorescent reporter cerulean, but it can be replaced by any other protein or RNA-encoding gene. For example, the GOI can be a miRNA or guide RNA that can regulate endogenous genes.

[0146] The present invention differs from the aforementioned TANGO and CHACHA systems in that these systems release transcription factors pre-fused with either a GPCR or a β-repressor protein, and these transcription factors accumulate over time. In contrast, in the present invention, all elements remain functional after one action of the protein-protein interaction, thus exhibiting multiple conversions. The target gene regulated by the TANGO and the systems of the present invention is under the regulation of a chimeric promoter. In the ChaCha system, appropriately designed gRNAs can also be used to regulate endogenous genes. Another difference between the present invention and the TANGO assay is that, in the present invention, in addition to the chimeric GPCR fusion and the β-repressor protein fusion, an additional component, namely RR, is required.

[0147] The HK domain, fused with GPCR and β-repressor protein, is only 223 aa in size. In TANGO, the fusion protein sizes are 240 aa and 341 aa, respectively. For ChaCha, these are 240 aa and 1900 aa, respectively, except that gRNA expression is required. Due to this smaller size, the system is easier to construct and places less burden on the cells.

[0148] The system of this invention comprises two signal amplification steps. The unique first step of this invention is generated by reconstructed HK, which phosphorylates multiple copies of RR. The second level of amplification, also present in the TANGO and ChaChaCha systems, is a gene-induced catalytic property of RR. Compared to the TANGO system, the two levels of amplification in this invention result in a 10-fold increase in dynamic range.

[0149] The system of this invention does not desensitize over time; the same protein can be reactivated after several cycles of ligand presence / absence. In contrast, the elements of the TANGO and ChaCha systems can only be used once, therefore their system activity depends on protein degradation and protein resynthesis, which are inherently slow processes. This characteristic allows the system of this invention to switch from an on state to an off state more quickly, and vice versa.

[0150] Because this invention incorporates an additional level of amplification compared to other methods, it can detect small amounts of protein and weak protein-protein interactions. An advantage of this feature is that the expression of the components required for the assay can be regulated from low to high within the cell. Thus, the expression levels of system components can be set to achieve a suitable ligand-inducible dynamic range. Therefore, this method reduces ligand-independent signaling that can occur due to protein overexpression in earlier methods.

[0151] Specifically, the measurements of this invention show a higher dynamic range than those of TANGO. Compared to TANGO, this parameter will facilitate the automated analysis of the results produced by this invention.

[0152] Another advantage of the system of this invention is that it can be multiplexed by using different HK-RR pairs simultaneously. Due to the vast number of natural two-component systems, its multiplexing potential is enormous. Each chimeric pair will be independent and induce different outputs. Therefore, in the same experiment, one will be able to test the effects of compounds on multiple protein-protein interactions or multiple GPCRs simultaneously. Multiplexing by other methods is more difficult because of the limited number of well-characterized TEV proteases.

[0153] The system of this invention also achieves reversibility due to the spontaneous dephosphorylation of RR. In the absence of interaction between the protein pairs, the kinase is no longer active and does not phosphorylate RR. Unphosphorylated RR cannot induce GOI expression. In the cases of TANGO and ChaCha, system reversibility is difficult because it requires the time-consuming degradation of the transcriptional activator released following the interaction between the GPCR and the β-repressor protein.

[0154] Finally, the assays of this invention are highly modular. As in the case of GPCR-induced assays, the same pair of complementary HK fragments can be used to detect protein-protein interactions in the cytoplasm and protein-protein interactions localized to the membrane. Other assays require significant tweaking to probe different types of protein-protein interactions, and, for example, the TANGO assay is specific to the GPCR pathway and has not yet been used to probe general protein-protein interactions.

[0155] This invention can have a variety of applications.

[0156] 1) It can be used to develop new screening assays to identify compounds that interact with GPCRs. The resulting assay is expected to have better specificity and a higher dynamic range than existing assays. It will also be easier to multiplex. The commercialization of this assay can be achieved by selling stable cell lines containing GPCRs that fuse with HK, similar to what DiscoverX and ThermoFischer (PathHunter and TANGO) are currently doing.

[0157] 2) It can be used to screen compounds that regulate protein-protein interactions and thus for drug discovery. Unlike yeast 2-hybrids, the screening assay can be performed in mammalian cells, a more relevant system. Furthermore, for the present invention, the same assay can be used for proteins located in different cellular compartments (nucleus, cytoplasm, or membrane).

[0158] 3) Existing cell-based therapeutics typically utilize signaling pathways involving protein-protein interactions on the cytoplasmic side of the membrane. This includes CAR-T cells, where the binding of antigens to extracellular antibody fragments recruits protein-interacting couplers; these interactions can then be rearranged using the inventors' method to produce a therapeutic effect. Example

[0159] It is known that the intracellular cytoplasmic domains of HK can dimerize and autophosphorylate.

[0160] This invention addresses the question of whether certain cytoplasmic domains possess reduced intrinsic signal transduction capabilities. To this end, the inventors conducted stepwise truncation mutagenesis on HKs to identify domains that cannot self-dimerize. Figure 1 b). The first group of truncated mutants contains the entire cytoplasmic domains of EnvZ and NarX (EnvZ and NarX, respectively). 180-450 and NarX 176-598 The second group is characterized by a deeper truncation, with the N-terminus located approximately 20 amino acids (aa) upstream of the histidine (EnvZ respectively). 223-450 and NarX 379-598 The inventors discovered that an EnvZ truncated mutant co-expressed with the homologous response regulator OmpR in HEK293 cells constitutively signals and induces the expression of the OmpR-regulated reporter mCerulean at levels comparable to wild-type EnvZ. Figure 1c). This result is consistent with the fact that the small EnvZ domain containing histidine (“domain A”, aa 223-289) is responsible for homodimerization. On the other hand, in the presence of homologous RR NarL and NarL inducible reporters, truncated mutants of NarX showed a size-dependent reduction in the basal signal, with the shortest mutant NarX... 379-598 Reaching background level ( Figure 1 d). There are several possible explanations for this result, including (1) decreased protein stability; (2) decreased kinase activity and / or increased phosphatase activity in the truncated mutant; and (3) the mutant's inability to self-dimerize. Of these explanations, only the last would support the eventual establishment of synthetic signal transduction. To examine whether forced dimerization could restore signal transduction, the inventors attempted to fuse a truncated NarX domain to a pair of proteins that form strong heterodimers in mammalian cells. The reason is that if only dimerization is impaired, these identical mutants, linked to the protein with strong affinity for each other, will dimerize and transduce downstream signals.

[0161] SynZip1 / SynZip2

[0162] The inventors used known interaction pairs SynZip1 and SynZip2 and fused them into the short NarX structural domain NarX. 379-598 The N- and C-termini. (Observed) Figure 6 Co-expression of SynZip1 and SynZip2 fusions at the N-terminus of the short NarX domain resulted in increased reporter expression, although the expression was much lower than that of the full-length NarX. On the other hand, co-expression of SynZip fusions at the C-terminus of NarX did not result in any dimerization-triggered increase in reporter expression. In summary, the results suggest the possibility of forced dimerization via fusion with the N-terminus of the short cytoplasmic NarX domain as a signaling mechanism, consistent with the position of the sensor domain relative to the cytoplasmic domain in the full-length HK. However, the quantitative performance was poor. Parallel to this study, the inventors optimized the promoters driving their constructs to ensure they were unresponsive to external stimuli and that expression levels were balanced. Figure 7 ).

[0163] For any synthetic signaling system, it is important to avoid nonspecific alterations to signal readout. In the system of this invention, the components are expressed by constitutive promoters. However, these promoters are actually controlled by highly expressed transcription factors such as Sp1, and there is always a risk that these factors are directly affected by external stimuli through unrelated endogenous pathways. This would lead to changes in constitutive expression and, consequently, noticeable changes in signal readout, which are unrelated to and unartefactual to the effects studied. To eliminate these confounding factors, the inventors examined the robustness of numerous constitutive promoters under various stimuli and compared the expression of iRFP from the CMV and EF1α promoters in the presence of different compounds commonly used to induce cell signaling (epinephrine and procaterol). In culture media without any of the compounds, the activities of the two promoters were similar. However, in the presence of epinephrine and procaterol, the activity of the CMV promoter was induced twofold, while the activity of the EF1α promoter was unaffected. Figure 7 a).

[0164] The activity of the NarX / NarL system expressed from the CMV, EF1α, and EF1α-V1 promoters was quantified to determine whether overexpression of the truncated HK cytoplasmic domain increased background levels and responded to nonspecific interactions. The EF1α-V1 promoter was approximately 5-fold weaker than EF1α. Results demonstrated that comparable reporter gene expression was obtained with NarX expressed from any of the tested promoters. Figure 7 b, comparing lanes 2 and 3 and lanes 5 and 6). However, when NarL was expressed from a weaker promoter, a decrease in reporter gene expression was observed (supplementary). Figure 2 b). Based on these results, the inventors used EF1α-V1 to drive NarX-derived constructs and EF1α to drive NarL expression.

[0165] The inventors further explored the possibility of optimizing this effect. It is known that HKs can be divided into two families in terms of autophosphorylation mechanism. In the "cis" family, the phosphoryl group is transferred from an ATP molecule bound to the CA domain of the same monomer to a histidine. In the "trans" family, the phosphoryl group is transferred from an ATP bound to one monomer to a phosphorylated histidine in another monomer. Figure 2 a). For all HKs, phosphoryl transfer can be terminated by mutating the ATP-binding site or histidine. In the case of "cis" family HKs, the heterodimer formed between the ATP-binding site mutant and the histidine mutant cannot transduce signals, but in the case of "trans" family HKs, it can still actually transmit signals from the histidine mutant monomer to the ATP-binding site mutant monomer via unidirectional phosphate transfer. Figure 2 b). Therefore, the mutants complement each other as the "trans"-family HKs.

[0166] The inventors hypothesize that dimerization between complementary mutants could lead to more efficient transduction due to reduced phosphatase activity of mutant HK toward its homologous response regulator. Using protein alignment, the inventors identified putative residues in the CA domain of NarX that are important for ATP binding.

[0167] Mutation analysis of the amino acids in the EnvZ CA domain identified asparagine at position 347 as the original source of EnvZ kinase activity. The CA domain of histidine kinase belongs to the large family of ATPase domains in HSP90 chaperone / DNA topoisomerase II / histidine kinase proteins (superfamily 55874, http: / / supfam.org / SUPERFAMILY / cgi-bin / scop.cgi?sunid=55874). To determine whether EnvZ's N347 is conserved in NarX, the inventors compared EnvZ and NarX with proteins containing the ATPase domain. The comparison identified Asn509 in NarX as a conserved residue, which may be important for ATP binding.

[0168] Because full-length HKs constitutively signal in mammalian cells, the inventors established a complementation assay in HEK293 cells by co-transfecting different combinations of codon-optimized NarX mutants with homologous downstream RR NarL and a reporter gene driven by the NarL-responsive promoter. In this analysis ( Figure 2 c) shows that (i) the full-length wild-type NarX can emit signals as previously indicated; (ii) as expected, mutants of the aspartic acid in the CA domain, which is important for ATP binding, or the histidine in the DHp domain, cannot emit signals independently; and (iii) co-expression of the NarX complementary mutant partially restores the signal to the level obtained by the wild-type NarX.

[0169] Next, the same mutation was introduced into the short cytoplasmic domain of NarX. Figure 2 d) The resulting mutant was then fused at the N-terminus to the SynZip1 and SynZip2 peptides, as previously done with the wild-type domain, to generate the construct SynZip1::NarX. 379-598 H399Q (from now on, for simplicity, it will be labeled SynZip1::H) mut ) and SynZip2::NarX 379 -598 N509A (marked as SynZip2::N) mut ()( Figure 2 e). The inventors also reversed the fusion pair to produce SynZip2::NarX.379- 598 H399Q(SynZip2:H) mut ) and SynZip1::NarX 379-598 N509A(SynZip1::N mut It was found that when these construct pairs were co-expressed with NarL RR in HEK293 cells in the presence of a NarL-responsive reporter, signaling via NarL was completely restored to the levels obtained with full-length wild-type NarX, and produced a much stronger signal than that of the wild-type short NarX fusion. Figure 8 For both pairs, the recovery is the same. Figure 2 f, Articles 11 and 12). Signal propagation stops when one or both of the fused SynZip domains are missing. Figure 2 f, Articles 4-8 Figure 9 This indicates that dimerization of the fusion domain is necessary and sufficient to recover signal transduction. Interestingly, all are fused to SynZip1(SynZip1::H mut and SynZip1::N mut Complementary NarX mutant pairs also lead to increased signal transduction activity. Figure 2 f, Article 9), is consistent with the (weaker) effect observed with wild-type domains under similar conditions. Figure 6 (Article 4). This leads to the hypothesis that the SynZip1 domain is capable of homodimerization, albeit with reduced affinity compared to the SynZip1-SynZip2 interaction. Indeed, studies in the original literature indicate that SynZip1 exists as both monomer and dimer in size exclusion chromatography experiments, and to a greater extent than SynZip2 alone. To evaluate the dose-response behavior of signal intensity, the inventors characterized the output levels of NarX-derived constructs at different plasmid doses ( Figure 2 g). Activity increased proportionally with plasmid dose, with full-length wild-type NarX showing the highest dose sensitivity, likely reflecting the strongest dimerization constant. SynZip1-SynZip2 showed a slightly reduced but comparable dimerization behavior, and SynZip1 clearly showed poorer dimerization. For example, a reduction of approximately 16-fold in plasmid amount compared to the initially used conditions (6.25 ng instead of 100 ng) reduced SynZip1-SynZip1 signaling to background levels while still resulting in strong SynZip1-SynZip2 dimerization, consistent with expectations.

[0170] In summary, these experiments demonstrate that NarX-initiated signal transduction can be restored upon forced dimerization of complementary truncated mutant domains, and that this restoration is dose- and interaction strength-dependent. Consistent with current knowledge of the two-component signal stoichiometry in *E. coli* with an HK:RR ratio of approximately 1:30, NarX expression yields approximately 10% of the fully activated reporter output compared to NarL. This is because the promoter driving NarL, EF1α-V1 (see Methods), is approximately 5-fold weaker than the wild-type EF1α promoter driving NarL, and furthermore, a 1:2 plasmid dose ratio (i.e., 50 ng NarX-derived plasmid vs. 100 ng RR-encoding plasmid) saturated the response.

[0171] FK506 / FKBP

[0172] For true signal transduction to occur, the dimerization of the NarX domain should preferably be controlled by an external stimulus. Inducible protein-protein interactions are common signal transduction mechanisms in the cytoplasm and transmembrane. In the presence of a small A / C heterodimer (rapamycin analog C16-(S)-7-methylindolerapamycin, also known as AP21967), signal transduction can occur via FK506-binding protein 12 (FKBP) and the FKBP12-rapamycin-binding domain (FRB) mutant FRB. T2098L Ligand-induced heterodimerization occurs between them in a fully characterized manner.

[0173] To find out whether the NarX domain can transduce this interaction ( Figure 3 a) The above-mentioned complementary histidine and asparagine NarX mutants were respectively coupled to FKBP (FK) and FRB at their N-terminus. T2098L (FR) protein fusion produces the fusion variant FK::NarX 379-598 H399Q(FK::H mut ) and FR::NarX 379-598 N509A(FR::N mut ), and the reverse of FR::H mut and FK::N mut First, it was confirmed that the A / C does not affect wild-type NarX / NarL systems, whether using NarX or NarL in the absence or presence of a 100nM A / C. 379-598 Transfected HEK cells ( Figure 3 b, paragraphs 2 and 3). Next, the inventors probed the ligand-induced signal in a manner similar to that used in the SynZip1-SynZip2 experiment, expressing different fusion variants and control constructs in HEK293 cells in the presence of the response regulator NarL and a NarL-activated reporter construct, this time with and without ligand ( Figure 3b, Figure 8 As expected, full-length wild-type NarX is constitutively active. In all cases other than wild-type NarX, no signal is generated in the absence of ligands. Strong signaling is induced only when (i) the NarX-derived domains contain complementary histidine and asparagine mutations and (ii) they are fused with FKBP and FRB interacting couplers, respectively. Figure 3 b, Articles 11 and 12). Then, the inventors continued to use FR::H mut and FK::N mut The dose-response behavior of this engineered signal transduction pathway was used to characterize the signal transduction pathway. Figure 3 c). The dose-response showed the expected Hill function dependence, with the EC50 determined to be 1.3 nM in the experimental context, compared to the published values ​​of 10 nM and 36 nM.

[0174] The above results demonstrate the ability of TCS-based components to mediate signal transduction in the cytoplasm. However, a large amount of signal transduction occurs across the entire membrane. Many transmembrane signaling pathways involve protein-protein interactions on the lipid bilayer cytoplasmic surface, including an important class of signaling pathways initiated by G protein-coupled receptors (GPCRs), a family of hundreds of proteins. A key step in GPCR signal transduction is the formation of a complex between the GPCR itself and the protein β-repressor, followed by various processes including GPCR internalization, recycling, and signal transduction. It has been previously shown that this interaction is sufficient to reconnect the GPCR signal through specific proteolytic cleavage by a fusion transcription activator. The inventors hypothesized that this interaction could also catalyze transmembrane signal transduction via a two-component pathway. To this end, the inventors used a truncated histidine mutant of NarX, NarX... 379-598 H399Q, fused to the GPCR ADRB2-AVPR2: a chimera of isopropylquinolone-activated adrenaline receptor β2 (ADRB2) and a cytoplasmic fragment of arginine vasopressin receptor 2 (AVPR2). The inventors also used a truncated asparagine mutant of NarX, NarX... 379-598 N509A, fused to β-repressor protein 2 ( Figure 4 a). Finally, the inventors reversed these fusions to check if the effects were symmetrical.

[0175] First, it was confirmed that propylquinolone does not affect signal transduction via the NarX / NarL system. In the absence or presence of 100 nM propylquinolone, NarL and NarL-activated reporters were used with NarX or NarX... 379-598 Co-transfected HEK293 cells showed reporter gene expression in complete form and in the background, independent of isopropylquinolone valerate. Figure 4b, paragraphs 2 and 3). In experiments combining complementary NarX mutants with GPCR receptors, β-repressor proteins, or both, respectively ( Figure 4 b、 Figure 11 a) It was found that there was a certain amount of β-inhibitory protein and a single GPCR receptor-independent signal transduction independent of propylquinolone. Figure 4 (b, strips 9 and 10). The effects of GPCRs were more pronounced, indicating that the receptor dimerized in a ligand-independent manner. Importantly, when the complementary NarX mutant was fused to both the GPCR receptor and the β-repressor protein, very strong propylquinolone-trimethoprim ... Figure 4 b, Sections 11 and 12). The dynamic range obtained using the two-component-based system is greater than that obtained using the TANGO assay (a GPCR activation assay based on proteolysis). Figure 11 b).

[0176] To determine whether the synthesized signal cascade could recapitulate the effects of different known GPCR ligands, the inventors characterized the presence of ADRB2-AVPR2::H mut and β-repressor protein::N mut Yes, systemic dose response was observed in the presence of two agonists (isoproquinolone and isoproterenol) and one partial agonist (clenbuterol). Two complete agonists, isoproquinolone (…), were observed. Figure 4 C, blue line) and isoproterenol ( Figure 4 C (red line) induces strong downstream gene expression in a dose-dependent manner, reaching the same maximum response at saturation doses. In the presence of the partial agonist clenbuterol, reporter gene expression is 3.5 times lower than in the presence of the full agonist. Figure 4 C, comparing the green line with the blue and red lines). Furthermore, single-cell flow cytometry data indicate unimodal rather than bimodal induction ( Figure 4 d, TCS data). The EC50 values ​​of isoproterenol, isoproterenol, and clenbuterol, determined from dose-response curves, were 5 nM, 30 nM, and 14 nM, respectively. These values ​​are similar to those described in the literature and determined by TANGO assays. Figure 4 c, dashed line, second axis). Note that although the TANGO assay resulted in higher absolute reporter expression, leakage was significantly higher compared to the TCS-based mechanism, and the single-cell data were bimodal. Figure 4 d, TANGO data). The inventors also characterized the effect of the antagonist propranolol in the presence of propylquinolone acetonide and found that in the signal cascade of the present invention, the antagonist inhibits the effect of propylquinolone acetonide in a dose-dependent manner ( Figure 4E, blue line). The inventors determined the IC50 of the antagonist in this experiment to be 2 nM, which is similar to the value obtained using TANGO ( Figure 4 e, dashed line). These results demonstrate that the system of the present invention can faithfully transduce a variety of known effects of agonists and antagonists on GPCR activity, and can be used to extract quantitative data on interaction parameters.

[0177] Overview

[0178] Implementing non-natural signal transduction patterns in cells, particularly mammalian cells, is highly desirable for the rational control of cellular behavior and ultimately for the engineering of novel cellular functions in basic research, biotechnology, and medicine. Two-component signal transduction is evolutionarily very different from vertebrate signal transduction, and to the inventors' knowledge, there is no single instance described in vertebrate cells of histidine-to-aspartate phosphorylation. The natural mechanism of TCS signal transduction in prokaryotes relies on ligand-induced conformational changes in the HK dimer within the membrane, but directly implementing this mechanism in mammalian cells is elusive. Instead, the inventors sought a different strategy to control signal transduction by switching between the dissociated and bound states of the HK cytoplasmic domain, achieving essentially the same end result. In the case shown by the inventors, this is accomplished through ligand-induced protein dimerization switching, where the protein is fused to histidine and asparagine mutants of the truncated cytoplasmic domain of HKNarX, respectively. Similar qualitative effects were observed when the wild-type truncated domain was used instead of the mutant. However, the quantitative behavior is poor, and more importantly, the results cannot distinguish between the dimerization of the two interacting mating bodies induced by the mutant and the homodimerization of one of the mating bodies, as seen in SynZip1 and GPCR. One reason for the reduced dynamic range may be the stronger phosphatase activity of the wild-type domain compared to the histidine mutant.

[0179] This method preserves many features of primitive prokaryotic signal transduction. It is an amplified, multi-turnover process in which a single NarX dimer can phosphorylate multiple copies of the response regulator NarL, which in turn can induce multiple transcription initiation events. The inventors hypothesize that the two-step amplification results in a significantly increased dynamic range compared to proteolytic cleavage-based methods. Furthermore, upon stimulus withdrawal, signal transduction ceases due to spontaneous dephosphorylation of RR; if rapid signal transduction silencing is desired, this can be facilitated by cleverly using the wild-type HK domain, which retains its full phosphatase activity. Given the rich diversity of the TCS pathway, multiplexing of synthetic signaling pathways is feasible using the above method. In summary, the results point to a new modality for sensing and signal transduction in mammalian cells, both in the cytoplasm and across the membrane.

[0180] method

[0181] Use standard molecular cloning techniques available to technicians.

[0182] Plasmid construction.

[0183] Plasmids were constructed using standard cloning techniques. All restriction enzymes used in this work were purchased from New England Biolabs (NEB). Q5 high-fidelity DNA polymerase (NEB) was used for fragment amplification. Single-stranded oligonucleotides were synthesized using Sigma-Aldrich. Digestion products or PCR fragments were purified using the GenElute Gel Extraction Kit or the GenElute PCR Clean Up Kit (Sigma-Aldrich). Ligation was performed using T4 DNA ligase (NEB) via temperature cycling for 140 cycles between 10°C for 30 s and 30°C for 30 s. Gibson assembly was performed as described below. 5 μl of ligation product or Gibson assembly product was transformed into chemically competent *E. coli* DH5α or *E. coli* TOP10 and plated on LB agar containing 100 μg / ml ampicillin. The resulting clones were directly screened by colony PCR (Dream Taq Green PCR Master Mix, Thermo Scientific). The inventors amplified single clones in LB Broth Miller Difco (BD) supplemented with ampicillin and purified their plasmid DNA using the GenElutePlasmid Miniprep Kit (Sigma-Aldrich). All obtained plasmids were sequenced using Sanger sequencing via Microsynth. Promega PureYield was used. TM DNA for mammalian transfection was obtained from 100 ml of liquid culture using a Plasmid Midiprep System (A2495). The recovered DNA was further purified using a Norgen Endotoxin Removal Kit Mini (Cat.#27700) or a Midi (Cat.#52200). Short cloning methods for each construct used in this work are described below.

[0184] Gibson assembly method

[0185] Gibson assembly was performed in a final volume of 10 μl by mixing the vector (0.018 pmol) and insert (0.09 pmol) in 1X Gibson assembly buffer (0.1 M Tris-HCl, pH 7.5, 0.01 M MgCl2, 0.2 mM dGTP, 0.2 mM dATP, 0.2 mM dTTP, 0.2 mM dCTP, 0.01 M DTT, 5% (w / v) PEG-8000, 1 mM NAD), with 0.04 units of T5 exonuclease (NEB), 0.25 units of Phusion DNA polymerase (NEB), and 40 units of Taq DNA ligase (NEB). The negative control of the Gibson assembly consisted only of the vector. Gibson assembly was performed at 50 °C for 1 hour.

[0186] Recombinant DNA cloning methods

[0187] OmpR_RE-Cerulean(pMZ1): The mCerulean coding sequence from EF1α-cerulean(pKH24) was digested with NotI and SmaI and cloned into plasmid OmpR_RE-amCyan(pJH008) digested with AfeI and PspOMI.

[0188] CMV-envZ N347A(pMZ37): The 5' and 3' fragments of envZ were amplified by PCR from plasmid CMV-envZ(pJH001) using PR3687 / PR3708 and PR3707 / PR3709. Primers were designed to introduce a mutation that replaced the codon encoding asparagine (N) at position 347 with the codon encoding alanine (A). PCR product and plasmid CMV-envZ(pJH001) digested with XhoI and PvuII were also described. 14 All of them are assembled using Gibson mix.

[0189] CMV-EnvZ 223-450 (pMZ123): The 3' fragment of envZ was amplified by PCR from plasmid CMV-envZ(pJH1) using PR4345 / PR4346. Primers were designed to amplify from codon 20 upstream of the codon encoding phosphorylated histidine at position 243 to the end of the gene, with an ATG sequence inserted before this amplified sequence. The PCR product and plasmid CMV-envZ(pJH001) digested with XhoI and AgeI were assembled using Gibson Mix.

[0190] CMV-NarX N509A (pMZ160): The 5' and 3' fragments of narX were amplified by PCR from plasmid CMV-narX (pJH002) using PR4122 / PR4541 and PR4346 / PR4542. Primers were designed to introduce a mutation that replaced the codon encoding asparagine (N) at position 509 with the codon encoding alanine (A). The PCR product and plasmid CMV-envZ (pJH001), digested with XhoI and AgeI, were assembled using Gibson mix.

[0191] CMV-narX 379-598 (pMZ163): Extracted from plasmid CMV-narX(pJH002) using PR4345 / PR4546. 14 PCR amplification of the 3' fragment of narX was performed. Primers were designed to amplify from the 20th codon upstream of the 399th codon encoding phosphorylated histidine to the end of the gene, with an ATG sequence inserted before the amplified sequence. The PCR product and plasmid CMV-envZ(pJH001) digested with XhoI and AgeI were assembled using Gibson Mix.

[0192] EF1α-V1-envZ-mCherry (pMZ194): EF1α-V1, as a shortened form of EF1α, was amplified by PCR from plasmid pRA114 (Altamura et al., manuscript in preparation) using PR4733 / PR4734. The promoter and plasmid EnvZ-GGGGS-mCherry (pEM01714), digested with PspOMI and AgeI, were assembled using Gibson Mix.

[0193] CMV-SynZip1::narX 379-598 (pMZ200): The inventors synthesized the gBlock sequence (gBlock264) encoding the SynZip1 and G4S linkers de novo via IDT. The sequence was obtained from plasmid CMV-narX using PR4346 / PR4747. 176-598 (JH01014) PCR amplification of NarX 379-598 The coding sequence was obtained. gBlock, PCR products, and plasmid CMV-envZ(pJH001) digested with AgeI and XhoI were assembled using Gibson Mix.

[0194] CMV-narX 379-598 ::SynZip1(pMZ202): The inventors de novo synthesized the gBlock sequence encoding the G4S linker and SynZip1(gBlock265) using IDT. Extracted from plasmid CMV-narX using PR4122 / PR4747.379-598 (pMZ163)PCR amplification of NarX 379-598 The coding sequence was obtained. gBlock, PCR products, and plasmid CMV-envZ(pJH001) digested with AgeI and XhoI were assembled using Gibson Mix.

[0195] CMV-SynZip2::narX 379-598 (pMZ206): The inventors de novo synthesized the gBlock sequence (gBlock269) encoding the SynZip2 and G4S linkers using IDT. The sequence was obtained from plasmid CMV-narX using PR4346 / PR4747. 176-598 (JH010) PCR amplification of NarX 379-598 The coding sequence was obtained. gBlock, PCR products, and plasmid CMV-envZ(pJH001) digested with AgeI and XhoI were assembled using Gibson Mix.

[0196] CMV-narX 379-598 ::SynZip1(pMZ208): The inventors de novo synthesized the gBlock sequence encoding the G4S linker and SynZip1(gBlock270) using IDT. Extracted from plasmid CMV-narX using PR4122 / PR4748. 379-598 (pMZ163)PCR amplification of NarX 379-598 The coding sequence was obtained. gBlock, PCR products, and plasmid CMV-envZ(pJH001) digested with AgeI and XhoI were assembled using Gibson Mix.

[0197] CMV-FRB T2098L::CBRC(pMZ211): Extracted from plasmid FRB::CBRC using PR4122 / PR4541 and PR4346 / PR4542 27 PCR amplification was performed on the 5' and 3' fragments of FRB containing CBRC. Primers were designed to introduce a mutation that replaced the codon encoding threonine (T) at position Both098th (relative to the full-length protein serine / threonine-protein kinase TOR1) with a codon encoding leucine (L). The PCR product and the BamHI and AgeI-digested plasmid FRB::CBRC were assembled using Gibson Mix.

[0198] CMV-FKBP::narX 379-598 (pMZ214): Extracted from plasmid CBRN::FKBP using PR4766 / PR4767. 27 PCR amplification of the FKBP sequence was performed. The sequence was extracted from plasmid CMV-narX using PR4346 / PR4771.176-598 (pJH010) PCR amplification of NarX 379-598 The coding sequence was determined. Primers were designed to insert (G4S)2 adapters between the amplified fragments. PCR products and plasmid CMV-envZ(pJH001), digested with AgeI and XhoI, were assembled using Gibson mix.

[0199] CMV-FRB T2098L::narX 379-598 (pMZ215): The sequence encoding FRB T2098L was amplified by PCR from plasmid CMV-FRB::CBRC(pMZ211) using PR4769 / PR4770. The sequence was amplified from plasmid CMV-narX using PR4346 / PR4771. 176-598 (pJH010) PCR amplification of NarX 379-598 The coding sequence was determined. Primers were designed to insert (G4S)2 adapters between the amplified fragments. PCR products and plasmid CMV-envZ(pJH001), digested with AgeI and XhoI, were assembled using Gibson mix.

[0200] NarL_RE-cerulean (pMZ219): The minimal effector element NarL_RE was formed by annealing primers PR4892 and PR4893. The annealed product and the plasmid OmpR_RE-Cerulean (pMZ1) digested with AscI and NdeI were both assembled using Gibsonmix.

[0201] EF1α-V1-SynZip1::narX 379-598 (pMZ221): Extracted from plasmid CMV-SynZip1::NarX using PR3687 / PR4971. 379-598 (pMZ200)PCR amplification of sequences encoding SynZip1, G4S adapter, and NarX379-383. PCR products and plasmid EF1α-V1-envZ-mCherry (pMZ194) digested with AgeI and XhoI were assembled using Gibson Mix.

[0202] EF1α-V1-SynZip2::narX 379-598 (pMZ222): Extracted from plasmid CMV-SynZip2::narX using PR3687 / PR4971. 379-598 (pMZ206)PCR amplification of sequences encoding SynZip2, G4S adapters, and NarX379-383. PCR products and plasmid EF1α-V1-envZ-mCherry (pMZ194) digested with AgeI and XhoI were assembled using Gibson Mix.

[0203] EF1α-V1-SynZip1::narX 379-598 H399Q(pMZ223): Extracted from plasmid CMV-SynZip1::narX using PR4971 / PR4973. 379-598 (pMZ200) PCR amplification of sequences encoding SynZip1 and G4S adapters. PCR amplification of sequences encoding NarX from plasmid CMV-narX H399Q(pEM014) using PR3687 / PR4972. 379-598 The sequence of H399Q was obtained. The PCR product and plasmid EF1α-V1-envZ-mCherry(pMZ194) digested with AgeI and XhoI were assembled using Gibson Mix.

[0204] EF1α-V1-SynZip2::narX 379-598 H399Q(pMZ224): Extracted from plasmid CMV-SynZip2::narX using PR4971 / PR4973. 379-598 (pMZ206)PCR amplification of sequences encoding SynZip2 and G4S adapters. PCR amplification of sequences encoding NarX from plasmid CMV-narX H399Q(pEM014) using PR3687 / PR4972. 379-598 The sequence of H399Q was obtained. The PCR product and plasmid EF1α-V1-envZ-mCherry(pMZ194) digested with AgeI and XhoI were assembled using Gibson Mix.

[0205] EF1α-V1-SynZip1::narX 379-598 N509A(pMZ225): Extracted from plasmid CMV-SynZip1::narX using PR4971 / PR4973. 379-598 (pMZ200)PCR amplification of the sequence encoding SynZip1. PR3687 / PR4972 was used to amplify the sequence encoding NarX from plasmid CMV-narX N509A (pMZ160) PCR. 379-598 The sequence of N509A. PCR products and plasmid EF1α-V1-envZ-mCherry(pMZ194) digested with AgeI and XhoI were assembled using Gibson Mix.

[0206] EF1α-V1-SynZip2::narX 379-598 N509A(pMZ226): Extracted from plasmid CMV-SynZip2::narX using PR4971 / PR4973. 379-598The coding sequence for SynZip2 was amplified by (pMZ206) PCR. The coding sequence for NarX was amplified by PCR from CMV-narXN509A (pMZ160) using PR3687 / PR4972. 379-598 The sequence of N509A. PCR products and plasmid EF1α-V1-envZ-mCherry(pMZ194) digested with AgeI and XhoI were assembled using Gibson Mix.

[0207] EF1α-V1-FKBP::narX 379-598 H399Q(pMZ229): Extracted from plasmid CMV-FKBP::NarX using PR4974 / PR4973. 379-598 (pMZ214)PCR amplification of sequences encoding FKBP and (G4S)2 adapters. PCR amplification of sequences encoding NarX from plasmid CMV-narX H399Q(pEM014) using PR3687 / PR4972. 379-598 The sequence of H399Q was obtained. The PCR product and plasmid EF1α-V1-envZ-mCherry(pMZ194) digested with AgeI and XhoI were assembled using Gibson Mix.

[0208] EF1α-V1-FRB T2098L::narX 379-598 H399Q(pMZ230): Extracted from plasmid CMV-FRB T2098L::narX using PR4975 / PR4973. 379-598 (pMZ215)PCR amplification of the sequence encoding FRB T2098L and (G4S)2 adapter. PCR amplification of the sequence encoding NarX from plasmid CMV-narX H399Q(pEM014) using PR3687 / PR4972. 379-598 The sequence of H399Q was obtained. The PCR product and plasmid EF1α-V1-envZ-mCherry(pMZ194) digested with AgeI and XhoI were assembled using Gibson Mix.

[0209] EF1α-V1-FKBP::narX 379-598 N509A(pMZ231): Extracted from plasmid CMV-FKBP::narX using PR4974 / PR4973. 379-598 (pMZ214)PCR amplification of sequences encoding FKBP and (G4S)2 adapters. PCR amplification of sequences encoding NarX from plasmid CMV-narX N509A (pMZ160) using PR3687 / PR4972. 379-598The sequence of N509A. PCR products and plasmid EF1α-V1-envZ-mCherry(pMZ194) digested with AgeI and XhoI were assembled using Gibson Mix.

[0210] EF1α-V1-FRB T2098L::narX 379-598 N509A(pMZ232): Extracted from plasmid CMV-FRB T2098L::narX using PR4975 / PR4973. 379-598 (pMZ215)PCR amplification of the sequence encoding FRB T2098L and (G4S)2 adapter. PCR amplification of the sequence encoding NarX from plasmid CMV-narX N509A (pMZ160) using PR3687 / PR4972. 379-598 The sequence of N509A. PCR products and plasmid EF1α-V1-envZ-mCherry(pMZ194) digested with AgeI and XhoI were assembled using Gibson Mix.

[0211] EF1α-V1-narX(pMZ239): Extracted from plasmid CMV-narX(pJH002) using PR3687 / PR4979 14 The sequence encoding NarX was amplified by PCR. The PCR product and plasmid EF1α-V1-envZ-mCherry (pMZ194) digested with AgeI and XhoI were assembled using Gibson Mix.

[0212] EF1α-V1-narX 379-598 (pMZ241): The 3' fragment of narX was amplified by PCR from plasmid CMV-narX(pJH002) using PR4977 / PR3687. Primers were designed to amplify the sequence from codon 20 upstream of the codon encoding phosphorylated histidine at position 399 to the end of the gene, with an ATG sequence inserted before this amplified sequence. The PCR product and plasmid EF1α-V1-envZ-mCherry(pMZ194) digested with AgeI and XhoI were assembled using Gibson Mix.

[0213] EF1α-V1-narX H399Q(pMZ242): Extracted from plasmid CMV-narX H399Q(pEM014) using PR3687 / PR4979 14 The coding sequence of NarX was amplified by PCR. The PCR product and plasmid EF1α-V1-envZ-mCherry (pMZ194) digested with AgeI and XhoI were assembled using Gibson Mix.

[0214] EF1α-V1-narX 379-598 H399Q(pMZ244): The 3' fragment of narX was amplified by PCR from plasmid CMV-narX H399Q(pEM014) using PR4977 / PR3687. Primers were designed to amplify the sequence from codon 20 upstream of the codon encoding phosphorylated histidine at position 399 to the end of the gene, with an ATG sequence inserted before this amplified sequence. The PCR product and plasmid EF1α-V1-envZ-mCherry(pMZ194), digested with AgeI and XhoI, were assembled using Gibson Mix.

[0215] EF1α-V1-narX N509A (pMZ245): The sequence encoding NarX was amplified by PCR from plasmid CMV-narX N509A (pMZ160) using PR3687 / PR4979. The PCR product and plasmid EF1α-V1-envZ-mCherry (pMZ194) digested with AgeI and XhoI were assembled using Gibson Mix.

[0216] EF1α-V1-narX 379-598 N509A (pMZ247): The 3' fragment of narX was amplified by PCR from plasmid CMV-narX N509A (pMZ160) using PR4977 / PR3687. Primers were designed to amplify the sequence from codon 20 upstream of the codon encoding phosphorylated histidine at position 399 to the end of the gene, with an ATG sequence inserted before this amplified sequence. The PCR product and plasmid EF1α-V1-envZ-mCherry (pMZ194), digested with AgeI and XhoI, were assembled using Gibson Mix.

[0217] EF1α-narL(pMZ248): The EF1α promoter was amplified by PCR from plasmid pRA58 (Altamura et al., manuscript in preparation) using PR4732 / PR4978. The PCR product and plasmid CMV-narL(pJH004) digested with PspOMI and AgeI were assembled using GibsonMix.

[0218] EF1α-V1-narL (pMZ249): The EF1α-V1 promoter was amplified by PCR from plasmid pRA114 (Altamura et al., manuscript in preparation) using PR4734 / PR4978. The PCR product and plasmid CMV-narL (pJH004) digested with PspOMI and AgeI were assembled using Gibson Mix.

[0219] EF1α-V1-ARRB2::narX379-598 (pMZ250): The sequence encoding ARRB2 was amplified by PCR from plasmid CMV-ARRB2::TEV protease (pBH302) using PR4980 / PR4981. The sequence encoding NarX was amplified by PCR from plasmid CMV-narX (pJH2) using PR3687 / PR4982. 379-598 The sequence was determined. Primers were designed to insert G4S adapters between the amplified fragments. The PCR product and plasmid EF1α-V1-envZ-mCherry(pMZ194) digested with AgeI and XhoI were assembled using Gibson Mix.

[0220] EF1α-V1-ARRB2::narX 379-598 H399Q(pMZ251): The sequence encoding ARRB2 was amplified by PCR from plasmid CMV-ARRB2::TEV protease (pBH302) using PR4980 / PR4981. The sequence encoding NarX was amplified by PCR from plasmid CMV-narXH399Q(pEM014) using PR3687 / PR4982. 379-598 The sequence of H399Q was determined. Primers were designed to insert G4S adapters between the amplified fragments. PCR products and plasmid EF1α-V1-envZ-mCherry(pMZ194) digested with AgeI and XhoI were assembled using GibsonMix.

[0221] EF1α-V1-ARRB2::narX 379-598 N509A (pMZ252): The sequence encoding ARRB2 was amplified by PCR from plasmid CMV-ARRB2::TEV protease (pBH302) using PR4980 / PR4981. The sequence encoding NarX was amplified by PCR from plasmid CMV-narXN509A (pMZ160) using PR3687 / PR4972. 379-598 The sequence of N509A was determined. Primers were designed to insert G4S adapters between the amplified fragments. PCR products and plasmid EF1α-V1-envZ-mCherry(pMZ194) digested with AgeI and XhoI were assembled using GibsonMix.

[0222] EF1α-V1-ADRB2 1-341 ::AVPR2 343-371 ::narX 379-598 H399Q(pMZ257): Extracted from plasmid CMV-ADRB2 using PR4983 / PR4985 1-341 ::AVPR2 343-371 ::tTA(pBH312) PCR amplification encodes ADRB2 1-341 ::AVPR2343-371 The sequence was amplified by PCR from plasmid CMV-narX H399Q(pEM014) using PR3687 / PR4982, encoding NarX. 379-598 The sequence of H399Q was determined. Primers were designed to insert G4S adapters between the amplified fragments. PCR products and plasmid EF1α-V1-envZ-mCherry(pMZ194) digested with AgeI and XhoI were assembled using Gibson Mix.

[0223] EF1α-V1-ADRB2 1-341 ::AVPR2 343-371 ::narX 379-598 N509A(pMZ258): Extracted from plasmid CMV-ADRB2 using PR4983 / PR4985 1-341 ::AVPR2 343-371 ::tTA(pBH312) PCR amplification encodes ADRB2 1-341 The sequence ::AVPR2343-371 was obtained. The encoding NarX was amplified by PCR from plasmid CMV-narX N509A(pMZ160) using PR3687 / PR4972. 379-598 The sequence of N509A was determined. Primers were designed to insert G4S adapters between the amplified fragments. PCR products and plasmid EF1α-V1-envZ-mCherry(pMZ194) digested with AgeI and XhoI were assembled using Gibson Mix.

[0224] tTA_RE-cerulean(pMZ290): Extracted from plasmid tTA_RE-mCherry(pIM003) using PR5226 / PR5227. 12 The tTA-regulated promoter was amplified by PCR. The PCR product and plasmid DcuR_E-Cerulean (pMZ259) digested with AscI and AgeI were assembled using Gibson Mix.

[0225] EF1α-V1-ARRB2::TEV protease (pMZ291): ARRB2 encoded by EF1α-V1-ARRB2::TEV protease (pBH302) was amplified by PCR using PR5228 / PR5229. 283-409 The sequence was obtained from plasmid EF1α-V1-ARRB2::narX using PR5230 / PR5231. 379-598 (pMZ250) PCR amplification of the bGH poly(A) signal sequence. PCR product and plasmid EF1α-V1-ARRB2::narX digested with BsaI and AvrII. 379-598(pMZ250) was assembled using Gibson Mix.

[0226] EF1α::iRFP(pCS184): The iRFP coding sequence from CMV-iRFP(pCS12) was amplified by PR2258 / PR2259 PCR. The PCR product and plasmid EF1α::Citrine (pRA001, Altamura et al., manuscript in preparation) digested with BmtI and XbaI were assembled using a ligation mix.

[0227] EF1α-V1-ADRB2 1-341 ::AVPR2 343-371 ::tTA(pBH292): Extracted from plasmid CMV-ADRB2 using PR5232 / PR5233 1-341 ::AVPR2 343-371 ::tTA(pBH312) PCR amplification encodes ADRB2 254-341 AVPR2 343-371 The sequence of tTA. PCR product and plasmid EF1α-V1-ADRB2 digested with BsaI and XhoI. 1-341 ::AVPR2 343-371 ::narX 379-598 The H399Q(pMZ257) was assembled using Gibson Mix.

[0228] CMV-ARRB2::TEV protease (pBH302): The inventors synthesized de novo the gBlock sequence encoding a β-repressor protein-2 fused with TEV protease, consisting of 2 gBlocks (gBlock112 and gBlock 113), via IDT. The gBlocks and the plasmid pZsYellow1-N1 (Clontech 632445) digested with NotI and EcoRI were assembled using Gibson Mix.

[0229] CMV-OPRK1 1-345 ::AVPR2 343-371 ::tTA(pBH309): The inventors synthesized, de novo, the gBlock (gBlock114) sequence encoding KOR-1 fused with tTA and the gBlock (gBlock115) sequence encoding V2R via IDT. The KOR-1 encoding sequence from gBlock114 was amplified using PR2442 / PR2443 PCR. 1-345The sequence was obtained. The PCR product, gBlock115, and plasmid pZsYellow1-N1 (Clontech 632445) digested with XhoI and MfeI were assembled using Gibson Mix.

[0230] CMV-ADRB2 1-341 ::AVPR2 343-371 ::tTA(pBH312): The inventors synthesized the gBlock (gBlock118) sequence encoding the β-2 adrenergic receptor de novo via IDT. The sequence encoding ADRB2 from gBlock118 was amplified using PR2442 / PR2444 PCR. 1-341 The sequence. PCR product and plasmid CMV-OPRK1 digested with XhoI and BssHII. 1-345 ::AVPR2 343 -371 ::tTA(pBH309) is assembled using Gibson Mix.

[0231] CMV-narX 176-598 (pJH010): The 3' fragment of narX from CMV-narX (pJH002) was amplified using PR1021 / PR1023 PCR. Primers were designed to amplify the NarX sequence from the alanine-encoding codon at position 176 to the gene terminus, and an ATG sequence was inserted before this amplified sequence. The PCR product and CMV-narX (pJH002) were digested with XhoI and AgeI. The two digested products were then ligated together.

[0232] The following plasmids have been previously reported: CMV-envZ (pJH001), CMV-narX (pJH002), CMV-ompR (pJH003), CMV-narL (pJH004), OmpR_RE-AmCyan (pJH008), CMV-envZ_cyt (pJH009), CMV-envZ H243V (pEM013), CMV-narX H399Q (pEM014), and EnvZ-GGGGS-mCherry (pEM017) (Hansen, J. et al. Proc Natl Acad Sci USA 111, 15705-15710 (2014)). CBRN::FKBP and FRB::CBRC (Schramm, A. et al. Int J Mol Sci 19(2018)). Ef1α-mCerulean(pKH024), Ef1α-citrine(pKH025)Ef1α-mCherry(pKH026)and Junk-DNA(pBH265) (Prochazka, et al., Nat Commun 5, 4729(2014)). pTRE Bidirectional mCherry-pA(pIM003) (Angelici, B., et al., CellRep 16, 2525-2537(2016)). Plasmid CMV-iRFP(pCS12) was obtained from Addgene (plasmid 31857 (Filonov, G. Set al. Angewandte Chemie International Edition 51, 1448-1451(2012))).

[0233] Cell culture

[0234] The experiments in this work were performed on HEK293 (Cat#11631-017) purchased from Life Technologies. Cells were cultured at 37°C and 5% CO2 in DMEM (Gibco, Life Technologies; Cat#41966-052) supplemented with 10% FBS (Sigma-Aldrich; Cat#F9665) and 1% penicillin / streptomycin solution (Sigma-Aldrich; Cat#P4333). Cells were divided every 3–4 days using 0.25% trypsin-EDTA (Gibco, Life Technologies; Cat#25200-072). Cultures were propagated for up to two months before being replaced with fresh cell stock.

[0235] transfection

[0236] All transfections were performed using Lipofectamine 2000 transfection reagent (Life Technologies; Cat#11668027). All transfections were performed in 24-well plates (Thermo Scientific Nunc; NC-142475), with 400 ng of DNA transfected. 24 hours prior to transfection, cells were seeded at a density of 50,000 cells / well in 500 μl of DMEM. The plasmid for each sample was mixed as shown in Supplementary Table 3-18, and the volume was finalized to 50 μl with Opti-MEM I Reduced Serum (Gibco, Life Technologies Cat#31985-962). 1.5 μl of Lipofectamine 2000 was diluted in 50 μl of Opti-MEM I / sample to achieve a final reagent / μg DNA ratio of 3.75:1 μl reagent / μg DNA. After incubation for at least 5 minutes, the diluted Lipofectamine was added to the mixed DNA sample. The resulting mixture was gently vortexed and incubated at room temperature for 20 minutes before cell addition. Four hours after DNA addition, the culture medium was removed and replaced with 500 μl of fresh medium. When needed, 5 μl of the test chemical was added to the medium. The desired final concentrations of different 100X stock solutions were prepared as follows:

[0237] A / C heterodimer (Clontech; Cat#635057) stock solution was prepared in ethanol (Honeywell; Cat#02860) at the following concentrations: 250 μM, 50 μM, 20 μM, 8 μM, 3.2 μM, 1.28 μM, 512 nM, 205 nM, 81.9 nM, 32.8 nM, 13.1 nM, 5.24 nM, and 1.04 nM.

[0238] The stock solution of propylquinolone (Sigma; Cat#P9180-10MG) was prepared in DMSO (Sigma; Cat#D4540, BCBT0803) at concentrations of 1 mM, 286 μM, 81.6 μM, 23.3 μM, 10 μM, 6.6 μM, 1.9 μM, 544 nM, 155 nM, 44.4 nM and 12.7 nM.

[0239] Isoproterenol (Sigma; Cat#I6504) stock solutions were prepared in DMSO (Sigma; Cat#D4540) at concentrations of 1 mM, 286 μM, 81.6 μM, 23.3 μM, 6.6 μM, 1.9 μM, 544 nM, 155 nM, 44.4 nM, and 12.7 nM.

[0240] Clenbuterol (Sigma; Cat#C5423) stock solutions were prepared in DMSO (Sigma; Cat#D4540) at concentrations of 1 mM, 286 μM, 81.6 μM, 23.3 μM, 6.6 μM, 1.9 μM, 544 nM, 155 nM, 44.4 nM, and 12.7 nM.

[0241] Propranolol (Sigma; Cat#P0884) stock solution prepared in water (Invitrogen; Cat#10977-035): 1 mM, 286 μM, 81.6 μM, 23.3 μM, 6.6 μM, 1.9 μM, 544 nM, 155 nM, 44.4 nM and 12.7 nM.

[0242] Microscopic examination

[0243] Microscopic images were acquired 48 hours post-transfection. The inventors used a Nikon Eclipse Ti microscope equipped with a mechanized stage and a temperature-controlled chamber maintained at 37°C during image acquisition. Excitation light was generated by a Nikon Intensight C-HGFI mercury lamp and filtered through an optimized set of Semrock filter cubes. Images were acquired using a Hamamatsu, ORCA R2 camera with 10X objectives. Each Semrock cube consisted of an excitation filter, a dichroic mirror, and an emission filter. To minimize crosstalk between different fluorescent proteins, the inventors used the following setup: Cerulean: CFP HC (HC438 / 24, BS 458, HC 483 / 32), mCherry: TxRed HC (HC 624 / 40, BS 593, HC 562 / 40). For Cerulean and mCherry, images were acquired with 40ms exposures. Images were processed using ImageJ software for uniform contrast enhancement to improve visualization.

[0244] Flow cytometer

[0245] 48 hours after transfection, the culture medium was removed and the cells were inoculated with 200 μl Stem Pro. TM Accutase TMCells for FACS analysis were prepared by incubating at 37°C for 5 minutes with a cell dissociation reagent (Gibco, cat#A11105-01). After incubation, the plates were transferred to ice. To avoid potential cell damage, samples were prepared in continuous batches, ensuring that no single sample remained on ice for more than 1 hour. Prepared samples were measured using a BD LSR Fortessa II cell analyzer, combining excitation and emission to minimize crosstalk between different fluorescent reporters. Cerulean was measured using a 445 nm laser and a 473 / 10 nm emission filter, while mCherry was measured using a 561 nm excitation laser coupled with a 600 nm long-pass filter and a 610 / 20 emission filter. Cerulean and mCherry were measured at PMT voltages of 330 and 310, respectively, in all experiments. SPHERO Rainbow calibration particles (Spherotech; Cat#RCP-30-5A, BD) were used to ensure consistent device performance.

[0246] Data Analysis

[0247] Data analysis of histograms from general flow cytometry was performed using FlowJo software. In this work, fluorescence values ​​in the histograms, expressed in normalized expression units (Cerulean, norm.u.), were calculated as follows. Live cells were gated based on forward and lateral scattering readouts. Single cells in the population were gated according to their forward scattering area and forward scattering height. Cerulean-positive cells were gated based on negative controls, such that 99.9% of the cells in the control sample fell outside the selected gate. For each Cerulean-positive cell, the mean fluorescence intensity was calculated and multiplied by the frequency of the positive cells. This value was used as a measure of the total reporter signal in the sample and can be defined as the total intensity (TI). The TI of Cerulean was normalized by the TI of mCherry-positive cells (constitutive transfection control). Therefore, the relevant formula is: reporter intensity as shown in norm.u. [mean (reporter in reporter + reporter in cell) * frequency (reporter + cell)] / [mean (transfection marker in transfection marker + cell) * frequency (transfection marker + cell)].

[0248] sequence

[0249] In the event that the sequence given below differs from the sequence scheme submitted in text form herein, the following sequence shall prevail.

[0250] Table 1 Primer sequences

[0251]

[0252]

[0253]

[0254]

[0255] Table 2 List of synthetic DNA used for plasmid constructs

[0256]

[0257]

[0258]

[0259]

[0260] The table below lists the sequences of the synthetic promoters (underlined sequences indicate RR DNA binding sites, italic letters indicate TATA boxes. Start codons are shown in bold).

[0261]

[0262] Sequences of the CMV promoter, EF1α, and EF1α-V1

[0263] CMV promoter (SEQ ID 3)

[0264] gcgttgacattgattattgactagttattaatagtaatcaattacggggtcattagttcatagcccatatatggagttccgcgttacataacttacggtaaatggcccgcctggctgaccgcccaacgacccccgcccattgacgtcaataatgacgtatgttcccatagtaacgccaatagggactttccattgacgtcaatgggtggagtatttacggtaaactgcccacttggcagtacatcaagtgtatcatatgccaagtacgccccctattgacgtcaatgacggtaaatggcccgcctggcattatgcccagtacatgaccttatgggactttcctacttggcagtacatctacgtattagtcatcgctattaccatggtgatgcggttttggcagtacatcaatgggcgtggatagcggtttgactcacggggatttccaagtctccaccccattgacgtcaatgggagtttgttttggcaccaaaatcaacgggactttccaaaatgtcgtaacaactccgccccattgacgcaaatgggcggtaggcgtgtacggtgggaggtctatataagcagagctctctggctaact

[0265] >EF1α(SEQ ID 4)

[0266]

[0267] >EF1α-V1(SEQ ID 5)

[0268] ttaagctcgggcccTGGGCGGGATTCGTCTTGGGCGGGATCCTTGTCCACGTGATCGGGGGAGGGACTTTCCCGCTGGAGTGACTCATCTAGCCCACGTGATCTTCATGCCACGTGATCGATATGGGGACTTTCCTGACTCCCACGTGATCGCACCCCCACGTGATCCCGTAAGGGACTTTCCCTACTTTGCCTAGAGAAGGTGGCGCGGGGTAAACTGGGAAAGTGATGTCGTGTACTGGCTCCGCCTTTTTCCCGTGCTCAGGGGAGAACCGTATATAAGTGCAGTAGTCGCCGTGAACGTTCTTTTTCGCAACGTTAACTAGCACAGAACACAGGTAAGTGCCGTGTGTGGTTCCCGCGGGCGGCGACGGGGCCCGTGCCCACGTGATCAGGAGTTGGGCGGGATGTTATGAGTGACTCACGCCATCCACGTGATCTCAGACGGGACTTTCCATATTAAGTGACTCAGGATAAGGGACTTTCCCTACGGCCACGTGATCTCTTTTTGGGCGGGATGAGATTGGGACTTTCCTGTCCTGGGACTTTCCTACAGTTCAAACTCGACCACGTGATCTTATGACTGACGGGCGGGTGAGTCACCCACGGTGGCATGGGGGACTTTCCTTTAGGCGTTCATGTGACTCCACGGACAAGCCTCAGACAGTGGTTCAAAGTTTTTTTCTTCCATTTCAGGTGTCGTGAa

[0269] Sequence of narL:

[0270] >NarL(SEQ ID 6)

[0271] MSNQEPATILLIDDHPMLRTGVKQLISMAPDITVVGEASNGEQGIELAESLDPDLILLDLNMPGMNGLETLDKLREKSLSGRIVVFSVSNHEEDVVTALKRGADGYLL KDMEPEDLLKALHQAAAGEMVLSEALTPVLAASLRANRATTERDVNQLTPRERDILKLIAQGLPNKMIARRLDITESTVKVHVKHMLKKMKLKSRVEAAVWVHQERIF

[0272] VP48 sequence:

[0273] >VP48 (SEQ ID 7)

[0274] GPADALDDFDLDMLPADALDDFDLDMLPADALDDFDLDMLPG

[0275] EnvZ 180-450 EnvZ 223-450 NarX 176-598 and NarX 379-598 The amino acid sequences are underlined and italicized under phosphorylated histidines, and asparagine, which is important for the ATP-binding domain, is marked in bold and underlined.

[0276] >EnvZ 180-450 (SEQ ID 8)

[0277]

[0278] >EnvZ 223-450 (SEQ ID 9)

[0279]

[0280] NarX 176-598 (SEQ ID 10)

[0281]

[0282]

[0283] NarX 379-598 (SEQ ID 11)

[0284]

[0285] NarX 379-598 Mutant versions of aa are indicated by bold and underline.

[0286] NarX 379-598 (H399Q)(SEQ ID 12)

[0287]

[0288] NarX 379-598 (N509A)(SEQ ID 13)

[0289] sequence list <110> ETH Zurich <120> New methods for transducing protein-protein interactions <130> eth182wo <160> 79 <170> PatentIn version 3.5 <210> 1 <211> 134 <212> DNA <213> Artificial Sequence <220> <223> Synthetic promoter <400> 1 atttacattt tgaaacatct atagcgccgg catttacatt ttgaaacatc tatccatatg 60 ctctagaggg tatataatgg gggccactag tctactacca gagctcatcg ctagcgctac 120 cggtcgccac catg 134 <210> 2 <211> 124 <212> DNA <213> Artificial Sequence <220> <223> Synthetic promoter <400> 2 tacccctata ggggtatagc gccggctacc cctatagggg tatccatatg ctctagaggg 60 tatataatgg gggccactag tctactacca gagctcatcg ctagcgctac cggtcgccac 120 catg 124 <210> 3 <211> 601 <212> DNA <213> Cytomegalovirus <400> 3 gcgttgacat tgattattga ctagttatta atagtaatca attacggggt cattagttca 60 tagcccatat atggagttcc gcgttacata acttacggta aatggcccgc ctggctgacc 120 gcccaacgac ccccgcccat tgacgtcaat aatgacgtat gttcccatag taacgccaat 180 agggactttc cattgacgtc aatgggtgga gtatttacgg taaactgccc acttggcagt 240 acatcaagtg tatcatatgc caagtacgcc ccctattgac gtcaatgacg gtaaatggcc 300 cgcctggcat tatgcccagt acatgacctt atgggacttt cctacttggc agtacatcta 360 cgtattagtc atcgctatta ccatggtgat gcggttttgg cagtacatca atgggcgtgg 420 atagcggttt gactcacggg gatttccaag tctccacccc attgacgtca atgggagttt 480 gttttggcac caaaatcaac gggactttcc aaaatgtcgt aacaactccg ccccattgac 540 gcaaatgggc ggtaggcgtg tacggtggga ggtctatata agcagagctc tctggctaac 600 t 601 <210> 4 <211> 1182 <212> DNA <213> Homo sapiens <400> 4 gctccggtgc ccgtcagtgg gcagagcgca catcgcccac agtccccgag aagttggggg 60 gaggggtcgg caattgaacc ggtgcctaga gaaggtggcg cggggtaaac tgggaaagtg 120 atgtcgtgta ctggctccgc ctttttcccg agggtggggg agaaccgtat ataagtgcag 180 tagtcgccgt gaacgttctt tttcgcaacg ggtttgccgc cagaacacag gtaagtgccg 240 tgtgtggttc ccgcgggcct ggcctcttta cgggttatgg cccttgcgtg ccttgaatta 300 cttccacgcc cctggctgca gtacgtgatt cttgatcccg agcttcgggt tggaagtggg 360 tgggagagtt cgaggccttg cgcttaagga gccccttcgc ctcgtgcttg agttgaggcc 420 tggcctgggc gctggggccg ccgcgtgcga atctggtggc accttcgcgc ctgtctcgct 480 gctttcgata agtctctagc catttaaaat ttttgatgac ctgctgcgac gctttttttc 540 tggcaagata gtcttgtaaa tgcgggccaa gatctgcaca ctggtatttc ggtttttggg 600 gccgcgggcg gcgacggggc ccgtgcgtcc cagcgcacat gttcggcgag gcggggcctg 660 cgagcgcggc caccgagaat cggacggggg tagtctcaag ctggccggcc tgctctggtg 720 cctggcctcg cgccgccgtg tatcgccccg ccctgggcgg caaggctggc ccggtcggca 780 ccagttgcgt gagcggaaag atggccgctt cccggccctg ctgcagggag ctcaaaatgg 840 aggacgcggc gctcgggaga gcgggcgggt gagtcaccca cacaaaggaa aagggccttt 900 ccgtcctcag ccgtcgcttc atgtgactcc acggagtacc gggcgccgtc caggcacctc 960 gattagttct cgagcttttg gagtacgtcg tctttaggtt ggggggaggg gttttatgcg 1020 atggagtttc cccacactga gtgggtggag actgaagtta ggccagcttg gcacttgatg 1080 taattctcct tggaatttgc cctttttgag tttggatctt ggttcattct caagcctcag 1140 acagtggttc aaagtttttt tcttccattt caggtgtcgt ga 1182 <210> 5 <211> 705 <212> DNA <213> Homo sapiens <400> 5 ttaagctcgg gccctgggcg ggattcgtct tgggcgggat ccttgtccac gtgatcgggg 60 gagggacttt cccgctggag tgactcatct agcccacgtg atcttcatgc cacgtgatcg 120 atatggggac tttcctgact cccacgtgat cgcaccccca cgtgatcccg taagggactt 180 tccctacttt gcctagagaa ggtggcgcgg ggtaaactgg gaaagtgatg tcgtgtactg 240 gctccgcctt tttcccgtgc tcaggggaga accgtatata agtgcagtag tcgccgtgaa 300 cgttcttttt cgcaacgtta actagcacag aacacaggta agtgccgtgt gtggttcccg 360 cgggcggcga cggggcccgt gcccacgtga tcaggagttg ggcgggatgt tatgagtgac 420 tcacgccatc cacgtgatct cagacgggac tttccatatt aagtgactca ggataaggga 480 ctttccctac ggccacgtga tctctttttg ggcgggatga gattgggact ttcctgtcct 540 gggactttcc tacagttcaa actcgaccac gtgatcttat gactgacggg cgggtgagtc 600 acccacggtg gcatggggga ctttccttta ggcgttcatg tgactccacg gacaagcctc 660 agacagtggt tcaaagtttt tttcttccat ttcaggtgtc gtgaa 705 <210> 6 <211> 216 <212> PRT <213> Escherichia coli <400> 6 Met Ser Asn Gln Glu Pro Ala Thr Ile Leu Leu Ile Asp Asp His Pro 1 5 10 15 Met Leu Arg Thr Gly Val Lys Gln Leu Ile Ser Met Ala Pro Asp Ile 20 25 30 Thr Val Val Gly Glu Ala Ser Asn Gly Glu Gln Gly Ile Glu Leu Ala 35 40 45 Glu Ser Leu Asp Pro Asp Leu Ile Leu Leu Asp Leu Asn Met Pro Gly 50 55 60 Met Asn Gly Leu Glu Thr Leu Asp Lys Leu Arg Glu Lys Ser Leu Ser 65 70 75 80 Gly Arg Ile Val Val Phe Ser Val Ser Asn His Glu Glu Asp Val Val 85 90 95 Thr Ala Leu Lys Arg Gly Ala Asp Gly Tyr Leu Leu Lys Asp Met Glu 100 105 110 Pro Glu Asp Leu Leu Lys Ala Leu His Gln Ala Ala Ala Gly Glu Met 115 120 125 Val Leu Ser Glu Ala Leu Thr Pro Val Leu Ala Ala Ser Leu Arg Ala 130 135 140 Asn Arg Ala Thr Thr Glu Arg Asp Val Asn Gln Leu Thr Pro Arg Glu 145 150 155 160 Arg Asp Ile Leu Lys Leu Ile Ala Gln Gly Leu Pro Asn Lys Met Ile 165 170 175 Ala Arg Arg Leu Asp Ile Thr Glu Ser Thr Val Lys Val His Val Lys 180 185 190 His Met Leu Lys Lys Met Lys Leu Lys Ser Arg Val Glu Ala Ala Val 195 200 205 Trp Val His Gln Glu Arg Ile Phe 210 215 <210> 7 <211> 42 <212> PRT <213> Escherichia coli <400> 7 Gly Pro Ala Asp Ala Leu Asp Asp Phe Asp Leu Asp Met Leu Pro Ala 1 5 10 15 Asp Ala Leu Asp Asp Phe Asp Leu Asp Met Leu Pro Ala Asp Ala Leu 20 25 30 Asp Asp Phe Asp Leu Asp Met Leu Pro Gly 35 40 <210> 8 <211> 272 <212> PRT <213> Escherichia coli <400> 8 Met Arg Ile Gln Asn Arg Pro Leu Val Asp Leu Glu His Ala Ala Leu 1 5 10 15 Gln Val Gly Lys Gly Ile Ile Pro Pro Pro Leu Arg Glu Tyr Gly Ala 20 25 30 Ser Glu Val Arg Ser Val Thr Arg Ala Phe Asn His Met Ala Ala Gly 35 40 45 Val Lys Gln Leu Ala Asp Asp Arg Thr Leu Leu Met Ala Gly Val Ser 50 55 60 His Asp Leu Arg Thr Pro Leu Thr Arg Ile Arg Leu Ala Thr Glu Met 65 70 75 80 Met Ser Glu Gln Asp Gly Tyr Leu Ala Glu Ser Ile Asn Lys Asp Ile 85 90 95 Glu Glu Cys Asn Ala Ile Ile Glu Gln Phe Ile Asp Tyr Leu Arg Thr 100 105 110 Gly Gln Glu Met Pro Met Glu Met Ala Asp Leu Asn Ala Val Leu Gly 115 120 125 Glu Val Ile Ala Ala Glu Ser Gly Tyr Glu Arg Glu Ile Glu Thr Ala 130 135 140 Leu Tyr Pro Gly Ser Ile Glu Val Lys Met His Pro Leu Ser Ile Lys 145 150 155 160 Arg Ala Val Ala Asn Met Val Val Asn Ala Ala Arg Tyr Gly Asn Gly 165 170 175 Trp Ile Lys Val Ser Ser Gly Thr Glu Pro Asn Arg Ala Trp Phe Gln 180 185 190 Val Glu Asp Asp Gly Pro Gly Ile Ala Pro Glu Gln Arg Lys His Leu 195 200 205 Phe Gln Pro Phe Val Arg Gly Asp Ser Ala Arg Thr Ile Ser Gly Thr 210 215 220 Gly Leu Gly Leu Ala Ile Val Gln Arg Ile Val Asp Asn His Asn Gly 225 230 235 240 Met Leu Glu Leu Gly Thr Ser Glu Arg Gly Gly Leu Ser Ile Arg Ala 245 250 255 Trp Leu Pro Val Pro Val Thr Arg Ala Gln Gly Thr Thr Lys Glu Gly 260 265 270 <210> 9 <211> 228 <212> PRT <213> Escherichia coli <400> 9 Met Ala Ala Gly Val Lys Gln Leu Ala Asp Asp Arg Thr Leu Leu Met 1 5 10 15 Ala Gly Val Ser His Asp Leu Arg Thr Pro Leu Thr Arg Ile Arg Leu 20 25 30 Ala Thr Glu Met Met Ser Glu Gln Asp Gly Tyr Leu Ala Glu Ser Ile 35 40 45 Asn Lys Asp Ile Glu Glu Cys Asn Ala Ile Ile Glu Gln Phe Ile Asp 50 55 60 Tyr Leu Arg Thr Gly Gln Glu Met Pro Met Glu Met Ala Asp Leu Asn 65 70 75 80 Ala Val Leu Gly Glu Val Ile Ala Ala Glu Ser Gly Tyr Glu Arg Glu 85 90 95 Ile Glu Thr Ala Leu Tyr Pro Gly Ser Ile Glu Val Lys Met His Pro 100 105 110 Leu Ser Ile Lys Arg Ala Val Ala Asn Met Val Val Asn Ala Ala Arg 115 120 125 Tyr Gly Asn Gly Trp Ile Lys Val Ser Ser Gly Thr Glu Pro Asn Arg 130 135 140 Ala Trp Phe Gln Val Glu Asp Asp Gly Pro Gly Ile Ala Pro Glu Gln 145 150 155 160 Arg Lys His Leu Phe Gln Pro Phe Val Arg Gly Asp Ser Ala Arg Thr 165 170 175 Ile Ser Gly Thr Gly Leu Gly Leu Ala Ile Val Gln Arg Ile Val Asp 180 185 190 Asn His Asn Gly Met Leu Glu Leu Gly Thr Ser Glu Arg Gly Gly Leu 195 200 205 Ser Ile Arg Ala Trp Leu Pro Val Pro Val Thr Arg Ala Gln Gly Thr 210 215 220 Thr Lys Glu Gly 225 <210> 10 <211> 424 <212> PRT <213> Escherichia coli <400> 10 Met Ala Arg Leu Leu Gln Pro Trp Arg Gln Leu Leu Ala Met Ala Ser 1 5 10 15 Ala Val Ser His Arg Asp Phe Thr Gln Arg Ala Asn Ile Ser Gly Arg 20 25 30 Asn Glu Met Ala Met Leu Gly Thr Ala Leu Asn Asn Met Ser Ala Glu 35 40 45 Leu Ala Glu Ser Tyr Ala Val Leu Glu Gln Arg Val Gln Glu Lys Thr 50 55 60 Ala Gly Leu Glu His Lys Asn Gln Ile Leu Ser Phe Leu Trp Gln Ala 65 70 75 80 Asn Arg Arg Leu His Ser Arg Ala Pro Leu Cys Glu Arg Leu Ser Pro 85 90 95 Val Leu Asn Gly Leu Gln Asn Leu Thr Leu Leu Arg Asp Ile Glu Leu 100 105 110 Arg Val Tyr Asp Thr Asp Asp Glu Glu Asn His Gln Glu Phe Thr Cys 115 120 125 Gln Pro Asp Met Thr Cys Asp Asp Lys Gly Cys Gln Leu Cys Pro Arg 130 135 140 Gly Val Leu Pro Val Gly Asp Arg Gly Thr Thr Leu Lys Trp Arg Leu 145 150 155 160 Ala Asp Ser His Thr Gln Tyr Gly Ile Leu Leu Ala Thr Leu Pro Gln 165 170 175 Gly Arg His Leu Ser His Asp Gln Gln Gln Leu Val Asp Thr Leu Val 180 185 190 Glu Gln Leu Thr Ala Thr Leu Ala Leu Asp Arg His Gln Glu Arg Gln 195 200 205 Gln Gln Leu Ile Val Met Glu Glu Arg Ala Thr Ile Ala Arg Glu Leu 210 215 220 His Asp Ser Ile Ala Gln Ser Leu Ser Cys Met Lys Met Gln Val Ser 225 230 235 240 Cys Leu Gln Met Gln Gly Asp Ala Leu Pro Glu Ser Ser Arg Glu Leu 245 250 255 Leu Ser Gln Ile Arg Asn Glu Leu Asn Ala Ser Trp Ala Gln Leu Arg 260 265 270 Glu Leu Leu Thr Thr Phe Arg Leu Gln Leu Thr Glu Pro Gly Leu Arg 275 280 285 Pro Ala Leu Glu Ala Ser Cys Glu Glu Tyr Ser Ala Lys Phe Gly Phe 290 295 300 Pro Val Lys Leu Asp Tyr Gln Leu Pro Pro Arg Leu Val Pro Ser His 305 310 315 320 Gln Ala Ile His Leu Leu Gln Ile Ala Arg Glu Ala Leu Ser Asn Ala 325 330 335 Leu Lys His Ser Gln Ala Ser Glu Val Val Val Thr Val Ala Gln Asn 340 345 350 Asp Asn Gln Val Lys Leu Thr Val Gln Asp Asn Gly Cys Gly Val Pro 355 360 365 Glu Asn Ala Ile Arg Ser Asn His Tyr Gly Met Ile Ile Met Arg Asp 370 375 380 Arg Ala Gln Ser Leu Arg Gly Asp Cys Arg Val Arg Arg Arg Glu Ser 385 390 395 400 Gly Gly Thr Glu Val Val Val Thr Phe Ile Pro Glu Lys Thr Phe Thr 405 410 415 Asp Val Gln Gly Asp Thr His Glu 420 <210> 11 <211> 221 <212> PRT <213> Escherichia coli <400> 11 Met Gln Glu Arg Gln Gln Gln Leu Ile Val Met Glu Glu Arg Ala Thr 1 5 10 15 Ile Ala Arg Glu Leu His Asp Ser Ile Ala Gln Ser Leu Ser Cys Met 20 25 30 Lys Met Gln Val Ser Cys Leu Gln Met Gln Gly Asp Ala Leu Pro Glu 35 40 45 Ser Ser Arg Glu Leu Leu Ser Gln Ile Arg Asn Glu Leu Asn Ala Ser 50 55 60 Trp Ala Gln Leu Arg Glu Leu Leu Thr Thr Phe Arg Leu Gln Leu Thr 65 70 75 80 Glu Pro Gly Leu Arg Pro Ala Leu Glu Ala Ser Cys Glu Glu Tyr Ser 85 90 95 Ala Lys Phe Gly Phe Pro Val Lys Leu Asp Tyr Gln Leu Pro Pro Arg 100 105 110 Leu Val Pro Ser His Gln Ala Ile His Leu Leu Gln Ile Ala Arg Glu 115 120 125 Ala Leu Ser Asn Ala Leu Lys His Ser Gln Ala Ser Glu Val Val Val 130 135 140 Thr Val Ala Gln Asn Asp Asn Gln Val Lys Leu Thr Val Gln Asp Asn 145 150 155 160 Gly Cys Gly Val Pro Glu Asn Ala Ile Arg Ser Asn His Tyr Gly Met 165 170 175 Ile Ile Met Arg Asp Arg Ala Gln Ser Leu Arg Gly Asp Cys Arg Val 180 185 190 Arg Arg Arg Glu Ser Gly Gly Thr Glu Val Val Val Thr Phe Ile Pro 195 200 205 Glu Lys Thr Phe Thr Asp Val Gln Gly Asp Thr His Glu 210 215 220 <210> 12 <211> 221 <212> PRT <213> Escherichia coli <400> 12 Met Gln Glu Arg Gln Gln Gln Leu Ile Val Met Glu Glu Arg Ala Thr 1 5 10 15 Ile Ala Arg Glu Leu Gln Asp Ser Ile Ala Gln Ser Leu Ser Cys Met 20 25 30 Lys Met Gln Val Ser Cys Leu Gln Met Gln Gly Asp Ala Leu Pro Glu 35 40 45 Ser Ser Arg Glu Leu Leu Ser Gln Ile Arg Asn Glu Leu Asn Ala Ser 50 55 60 Trp Ala Gln Leu Arg Glu Leu Leu Thr Thr Phe Arg Leu Gln Leu Thr 65 70 75 80 Glu Pro Gly Leu Arg Pro Ala Leu Glu Ala Ser Cys Glu Glu Tyr Ser 85 90 95 Ala Lys Phe Gly Phe Pro Val Lys Leu Asp Tyr Gln Leu Pro Pro Arg 100 105 110 Leu Val Pro Ser His Gln Ala Ile His Leu Leu Gln Ile Ala Arg Glu 115 120 125 Ala Leu Ser Asn Ala Leu Lys His Ser Gln Ala Ser Glu Val Val Val 130 135 140 Thr Val Ala Gln Asn Asp Asn Gln Val Lys Leu Thr Val Gln Asp Asn 145 150 155 160 Gly Cys Gly Val Pro Glu Asn Ala Ile Arg Ser Asn His Tyr Gly Met 165 170 175 Ile Ile Met Arg Asp Arg Ala Gln Ser Leu Arg Gly Asp Cys Arg Val 180 185 190 Arg Arg Arg Glu Ser Gly Gly Thr Glu Val Val Val Thr Phe Ile Pro 195 200 205 Glu Lys Thr Phe Thr Asp Val Gln Gly Asp Thr His Glu 210 215 220 <210> 13 <211> 221 <212> PRT <213> Escherichia coli <400> 13 Met Gln Glu Arg Gln Gln Gln Leu Ile Val Met Glu Glu Arg Ala Thr 1 5 10 15 Ile Ala Arg Glu Leu His Asp Ser Ile Ala Gln Ser Leu Ser Cys Met 20 25 30 Lys Met Gln Val Ser Cys Leu Gln Met Gln Gly Asp Ala Leu Pro Glu 35 40 45 Ser Ser Arg Glu Leu Leu Ser Gln Ile Arg Asn Glu Leu Asn Ala Ser 50 55 60 Trp Ala Gln Leu Arg Glu Leu Leu Thr Thr Phe Arg Leu Gln Leu Thr 65 70 75 80 Glu Pro Gly Leu Arg Pro Ala Leu Glu Ala Ser Cys Glu Glu Tyr Ser 85 90 95 Ala Lys Phe Gly Phe Pro Val Lys Leu Asp Tyr Gln Leu Pro Pro Arg 100 105 110 Leu Val Pro Ser His Gln Ala Ile His Leu Leu Gln Ile Ala Arg Glu 115 120 125 Ala Leu Ser Ala Ala Leu Lys His Ser Gln Ala Ser Glu Val Val Val 130 135 140 Thr Val Ala Gln Asn Asp Asn Gln Val Lys Leu Thr Val Gln Asp Asn 145 150 155 160 Gly Cys Gly Val Pro Glu Asn Ala Ile Arg Ser Asn His Tyr Gly Met 165 170 175 Ile Ile Met Arg Asp Arg Ala Gln Ser Leu Arg Gly Asp Cys Arg Val 180 185 190 Arg Arg Arg Glu Ser Gly Gly Thr Glu Val Val Val Thr Phe Ile Pro 195 200 205 Glu Lys Thr Phe Thr Asp Val Gln Gly Asp Thr His Glu 210 215 220 <210> 14 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 14 gctgctgctc tcgagtcatt atcattcgtg agtgtc 36 <210> 15 <211> 43 <212> DNA <213> Artificial Sequence <220> <223> Primers <400> 15 gctgctaccg gtcgccacca tggcccgctt gctccagccg tgg 43 <210> 16 <211> 72 <212> DNA <213> Artificial Sequence <220> <223> Primers <400> 16 cgcgcctgat tacaaaactt taaaaagtgc tgtagcgccg gctgattaca aaactttaaa 60 aagtgctgtc ca 72 <210> 17 <211> 70 <212> DNA <213> Artificial Sequence <220> <223> Primers <400> 17 tatggacagc actttttaaa gttttgtaat cagccggcgc tacagcactt tttaaagttt 60 tgtaatcagg 70 <210> 18 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> Primers <400> 18 taagcggaat tcatcttggc tgaggaatct t 31 <210> 19 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> Primers <400> 19 gcgaattcta gactacttgt acagctcgtc c 31 <210> 20 <211> 39 <212> DNA <213> Artificial Sequence <220> <223> Primers <400> 20 aatgtgaagc tagcgccacc atggctgaag gatccgtcg 39 <210> twenty one <211> 35 <212> DNA <213> Artificial Sequence <220> <223> Primers <400> twenty one aatgtaatct agatcactct tccatcacgc cgatc 35 <210> twenty two <211> twenty two <212> DNA <213> Artificial Sequence <220> <223> Primers <400> twenty two gctagcgcta ccggactcag at 22 <210> twenty three <211> 43 <212> DNA <213> Artificial Sequence <220> <223> Primers <400> twenty three tgggtggggt gcgtccgcgc gcacagaagt cccggaaaca ccg 43 <210> twenty four <211> 45 <212> DNA <213> Artificial Sequence <220> <223> Primers <400> twenty four tgggtggggt gcgtccgcgc gcacacagaa gctcctggaa ggcaa 45 <210> 25 <211> twenty two <212> DNA <213> Artificial Sequence <220> <223> Primers <400> 25 ggcacagtcg aggctgattt tc 22 <210> 26 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Primers <400> 26 tggcagcggg cgtcaagcag 20 <210> 27 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> Primers <400> 27 atggtggtgg cagcggcgag gtatggcaac g 31 <210> 28 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> Primers <400> 28 ctcgccgctg ccaccaccat gttggcgacg 30 <210> 29 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> Primers <400> 29 gaaattaata cgactcacta taggggac 28 <210> 30 <211> 57 <212> DNA <213> Artificial Sequence <220> <223> Primers <400> 30 gaaattaata cgactcacta taggggaccg gtcgccacca tggcagcggg cgtcaag 57 <210> 31 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Primers <400> 31 cacagtcgag gctgattttc 20 <210> 32 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> Primers <400> 32 gtttgagagc tgccgagagt gcttctctcg cg 32 <210> 33 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> Primers <400> 33 agcactctcg gcagctctca aacatagcca gg 32 <210> 34 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> Primers <400> 34 cttccagtgc agcttcaatc agatttccca gtgtg 35 <210> 35 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> Primers <400> 35 tctgattgaa gctgcactgg aagctctggg ac 32 <210> 36 <211> 60 <212> DNA <213> Artificial Sequence <220> <223> Primers <400> 36 gaaattaata cgactcacta taggggaccg gtcgccacca tgcaagagcg gcagcagcag 60 <210> 37 <211> 42 <212> DNA <213> Artificial Sequence <220> <223> Primers <400> 37 gacggccagt cttaagctcg ggcccgctcc ggtgcccgtc ag 42 <210> 38 <211> 47 <212> DNA <213> Artificial Sequence <220> <223> Primers <400> 38 gaagtcgtcg catggtggcg accggttcac gacacctgaa atggaag 47 <210> 39 <211> 43 <212> DNA <213> Artificial Sequence <220> <223> Primers <400> 39 gacggccagt cttaagctcg ggccctgggc gggattcgtc ttg 43 <210> 40 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Primers <400> 40 caagagcggc agcagcag 18 <210> 41 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Primers <400> 41 ttcgtgagtg tcaccctgc 19 <210> 42 <211> 57 <212> DNA <213> Artificial Sequence <220> <223> Primers <400> 42 gaaattaata cgactcacta taggggaccg gtcgccacca tgggcgtgca ggtggag 57 <210> 43 <211> 49 <212> DNA <213> Artificial Sequence <220> <223> Primers <400> 43 cgccaccgcc tgaaccgcctccaccttcca gttttagaag ctccacatc 49 <210> 44 <211> 57 <212> DNA <213> Artificial Sequence <220> <223> Primers <400> 44 gaaattaata cgactcacta taggggaccg gtcgccacca tggtagccat cctctgg 57 <210> 45 <211> 46 <212> DNA <213> Artificial Sequence <220> <223> Primers <400> 45 cgccaccgcc tgaaccgcctccacctgata tccgtctgaa cacgtg 46 <210> 46 <211> 43 <212> DNA <213> Artificial Sequence <220> <223> Primers <400> 46 aggcggttca ggcggtggcg ggtcgcaaga gcggcagcag cag 43 <210> 47 <211> 52 <212> DNA <213> Artificial Sequence <220> <223> Primers <400> 47 cgcgcctacc cctatagggg tatagcgccg gctaccccta taggggtatc ca 52 <210> 48 <211> 50 <212> DNA <213> Artificial Sequence <220> <223> Primers <400> 48 tatggatacc cctatagggg tagccggcgc tataccccta taggggtagg 50 <210> 49 <211> 44 <212> DNA <213> Artificial Sequence <220> <223> Primers <400> 49 ttcttccatt tcaggtgtcg tgaaccggtc gccaccatgg gctc 44 <210> 50 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> Priomer <400> 50 catcgtcatg gaagagaggg cgactattgc 30 <210> 51 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> Primers <400> 51 gcaatagtcg ccctctcttc catgacgatg 30 <210> 52 <211> 44 <212> DNA <213> Artificial Sequence <220> <223> Primers <400> 52 ttcttccatt tcaggtgtcg tgaaccggtc gccaccatgg gcgt 44 <210> 53 <211> 44 <212> DNA <213> Artificial Sequence <220> <223> Primers <400> 53 ttcttccatt tcaggtgtcg tgaaccggtc gccaccatgg tagc 44 <210> 54 <211> 57 <212> DNA <213> Artificial Sequence <220> <223> Primers <400> 54 ttcttccatt tcaggtgtcg tgaaccggtc gccaccatgc aagagcggca gcagcag 57 <210> 55 <211> 39 <212> DNA <213> Artificial Sequence <220> <223> Primers <400> 55 cctgattgga catggtggcg accggttcac gacacctga 39 <210> 56 <211> 42 <212> DNA <213> Artificial Sequence <220> <223> Primers <400> 56 ttcttccatt tcaggtgtcg tgaaccggtc gccaccatgc tt 42 <210> 57 <211> 52 <212> DNA <213> Artificial Sequence <220> <223> Primers <400> 57 tccatttcag gtgtcgtgaa ccggtcgcca ccatggggga gaaacccggg ac 52 <210> 58 <211> 45 <212> DNA <213> Artificial Sequence <220> <223> Primers <400> 58 ctcttgcgag ccaccgccac cgcagagttg atcatcatag tcgtc 45 <210> 59 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> Primers <400> 59 ggtggcggtg gctcgcaaga gcggcagcag cag 33 <210> 60 <211> 50 <212> DNA <213> Artificial Sequence <220> <223> Primers <400> 60 tccatttcag gtgtcgtgaa ccggtcgcca ccatggggca acccgggaac 50 <210> 61 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Primers <400> 61 ctcttgcgag ccaccgccac ccgatgaagt gtccttggcc 40 <210> 62 <211> 38 <212> DNA <213> Artificial Sequence <220> <223> Primers <400> 62 attacgccaa gctacggggg cacctcgaca tactcgag 38 <210> 63 <211> 41 <212> DNA <213> Artificial Sequence <220> <223> Primers <400> 63 ccttgctcac catggtggcg aggtaccgag ctcgaaatct c 41 <210> 64 <211> twenty one <212> DNA <213> Artificial Sequence <220> <223> Primers <400> 64 accgggagaa gcggggtctc g 21 <210> 65 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Primers <400> 65 cagtcgaggc tgattttctc gctcaagcgt aatctggaac 40 <210> 66 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Primers <400> 66 gttccagatt acgcttgagc gagaaaatca gcctcgactg 40 <210> 67 <211> twenty one <212> DNA <213> Artificial Sequence <220> <223> Primers <400> 67 ccgggagctt tttgcaaaag c 21 <210> 68 <211> twenty two <212> DNA <213> Artificial Sequence <220> <223> Primers <400> 68 ggacggggca tggactccgc ag 22 <210> 69 <211> 56 <212> DNA <213> Artificial Sequence <220> <223> Primers <400> 69 gcacagtcga ggctgatttt ctcgagtcat tactacccac cgtactcgtc aattcc 56 <210> 70 <211> 996 <212> DNA <213> Artificial Sequence <220> <223> Synthetic DNA for plasmid constructs <400> 70 agatctcgag ctcaagcttc gaattcgcca ccatggggga gaaacccggg accagggtct 60 tcaagaagtc gagccctaac tgcaagctca ccgtgtactt gggcaagcgg gacttcgtag 120 atcacctgga caaagtggac cctgtagatg gcgtggtgct tgtggaccct gactacctga 180 aggaccgcaa agtgtttgtg accctcacct gcgccttccg ctatggccgt gaagacctgg 240 atgtgctggg cttgtccttc cgcaaagacc tgttcatcgc cacctaccag gccttccccc 300 cggtgcccaa cccaccccgg ccccccaccc gcctgcagga ccggctgctg aggaagctgg 360 gccagcatgc ccaccccttc ttcttcacca taccccagaa tcttccatgc tccgtcacac 420 tgcagccagg cccagaggat acaggaaagg cctgcggcgt agactttgag attcgagcct 480 tctgtgctaa atcactagaa gagaaaagcc acaaaaggaa ctctgtgcgg ctggtgatcc 540 gaaaggtgca gttcgccccg gagaaacccg gcccccagcc ttcagccgaa accacacgcc 600 acttcctcat gtctgaccgg tccctgcacc tcgaggcttc cctggacaag gagctgtact 660 accatgggga gcccctcaat gtaaatgtcc acgtcaccaa caactccacc aagaccgtca 720 agaagatcaa agtctctgtg agacagtacg ccgacatctg cctcttcagc accgcccagt 780 acaagtgtcc tgtggctcaa ctcgaacaag atgaccaggt atctcccagc tccacattct 840 gtaaggtgta caccataacc ccactgctca gcgacaaccg ggagaagcgg ggtctcgccc 900 tggatgggaa actcaagcac gaggacacca acctggcttc cagcaccatc gtgaaggagg 960 gtgccaacaa ggaggtgctg ggaatcctgg tgtcct 996 <210> 71 <211> 1066 <212> DNA <213> Artificial Sequence <220> <223> Synthetic DNA for plasmid constructs <400> 71 gctgggaatc ctggtgtcct acagggtcaa ggtgaagctg gtggtgtctc gaggcgggga 60 tgtctctgtg gagctgcctt ttgttcttat gcaccccaag ccccacgacc acatccccct 120 ccccagaccc cagtcagccg ctccggagac agatgtccct gtggacacca acctcattga 180 atttgatacc aactagcca cagatgatga cattgtgttt gaggacttg cccggcttcg 240 gctgaagggg atgaaggatg acgactatga tgatcactc tgcggatcca gcttgtttaa 300 gggaccacgt gattacaacc cgatatcgag caccattgt catttgacga atgaatctga 360 tgggcacaca acatcgttgt atggtattgg atttggtcc ttcatcatta caacaagca 420 cttgtttaga agaaataatg gaacactgtt ggtccaatca ctacatggtg tattcaggt 480 caacaacc acgactttgc aaaacacct cattgatggg agggacatga taattattcg 540 catgcctaag gatttcccac cattcctca aaagctgaaa tttagagagc cacaaaggga 600 agagcgcata tgtcttgtga caacaactt ccaactaag agcatgtcta gcatggtgtc 660 agacactagt tgcacattcc cttcatctga tggcatattc tggagcatt ggattcaac 720 caagtggg cagtgtggca gtccattagt atcactaga gatgggttca ttgttggtat 780 acactcagca tcgaatttca ccacacaaa cattattc acagcgtgc cgaaaaactt 840 catggaattg ttgacaatc aggaggcgca gcagtgggtt agtggttggc gattaaatgc 900 tgactcagta ttgtgggggg gccataaagt tttcatgagc aaacctgaag agccttttca 960 gccagttaag gaagcgactc aactcatgaa tgaattggtg tactcgcaat acccatacga 1020 tgttccagat tacgcttgag cggccgcgac tctagatcat aatcag 1066 <210> 72 <211> 1201 <212> DNA <213> Artificial Sequence <220> <223> Synthetic DNA for plasmid constructs <400> 72 gcgctaccgg actcagatct cgaggccacc atggactccc cgatccagat cttccgcggg 60 gagccgggcc ctacctgcgc cccgagcgcc tgcctgcccc ccaacagcag cgcctggttt 120 cccggctggg ccgagcccga cagcaacggc agcgccggct cggaggacgc gcagctggag 180 cccgcgcaca tctccccggc catcccggtc atcatcacgg cggtctactc cgtagtgttc 240 gtcgtgggct tggtgggcaa ctcgctggtc atgttcgtga tcatccgata cacaaagatg 300 aagacagcaa ccaacattta catatttaac ctggctttgg cagatgcttt agttactaca 360 accatgccct ttcagagtac ggtctacttg atgaattcct ggccttttgg ggatgtgctg 420 480. tgcaagatag taatttccat tgattactac aacatgttca ccagcatctt caccttgacc atgatgagcg tggaccgcta cattgccgtg tgccaccccg tgaaggcttt ggacttccgc 540 acacccttga aggcaaagat catcaatatc tgcatctggc tgctgtcgtc atctgttggc atctctgcaa tagtccttgg aggcaccaaa gtcagggaag acgtcgatgt cattgagtgc tccttgcagt tcccagatga tgactactcc tggtgggacc tcttcatgaa gatctgcgtc 720 ttcatctttg ccttcgtgat ccctgtcctc atcatcatcg tctgctacac cctgatgatc 780 ctgcgtctca agagcgtccg gctccttct ggctcccgag agaaagatcg caacctgcgt 840 aggatcacca gactggtcct ggtggtggtg gcagtcttcg tcgtctgctg gactcccatt 900 cacatattca tcctggtgga ggctctgggg agcaccc acagcacagc tgctctctcc 960 agctattact tctgcatcgc cttaggctat accaacagta gcctgaatcc cattctctac gcctttcttg atgaaaactt caagcggtgt ttccgggact tctgttttcc actgaagatg aggatggagc ggcagagcac tagcagagtc cgaaatacag ttcaggaccc tgcttacctg agggacatcg atgggatgaa taaaccagta tgacaattgt tgttgttaac ttgtttattg 1200 c 1201 <210> 73 <211> 1167 <212> DNA <213> Artificial Sequence <220> <223> Synthetic DNA for plasmid constructs <400> 73 agcggtgttt ccgggacttc tgtgcgcgcg gacgcacccc acccagcctg ggtccccaag 60 atgagtcctg caccaccgcc agctcctccc tggccaagga cacttcatcg ggatccgaga 120 atctgtactt tcagctgaga ttagataaaa gtaaagtgat taacagcgca ttagagctgc 180 ttaatgaggt cggaatcgaa ggtttaacaa cccgtaaact cgcccagaag ctaggtgtag 240 agcagcctac attgtattgg catgtaaaaa ataagcgggc tttgctcgac gccttagcca 300 ttgagatgtt agataggcac catactcact tttgcccttt agaaggggaa agctggcaag 360 attttttacg taataacgct aaaagtttta gatgtgcttt actaagtcat cgcgatggag 420 caaaagtaca tttaggtaca cggcctacag aaaaacagta tgaaactctc gaaaatcaat 480 tagccttttt atgccaacaa ggtttttcac tagagaatgc attatatgca ctcagcgctg 540 tggggcattt tactttaggt tgcgtattgg aagatcaaga gcatcaagtc gctaaagaag 600 aaagggaaac acctactact gatagtatgc cgccattatt acgacaagct atcgaattat 660 ttgatcacca aggtgcagag ccagccttct tattcggcct tgaattgatc atatgcggat 720 tagaaaaaca acttaaatgt gaaagtgggt ccgcgtacag ccgggcgcgt acgaaaaaca 780 attacgggtc taccatcgag ggcctgctcg atctcccgga cgacgacgcc cccgaagagg 840 cggggctggc ggctccgcgc ctgtcctttc tccccgcggg acacacgcgc agactgtcga 900 cggccccccc gaccgatgtc agcctggggg acgagctcca cttagacggc gaggacgtgg 960 cgatggcgca tgccgacgcg ctagacgatt tcgatctgga catgttgggg gacggggatt 1020 ccccgggtcc gggatttacc ccccacgact ccgcccccta cggcgctctg gatatggccg 1080 acttcgagtt tgagcagatg tttaccgatg cccttggaat tgacgagtac ggtgggtagc 1140 aattgttgtt gttaacttgt ttattgc 1167 <210> 74 <211> 1304 <212> DNA <213> Artificial Sequence <220> <223> Synthetic DNA for plasmid constructs <400> 74 gctagcgcta ccggactcag atctcgaggc caccatgggg caacccggga acggcagcgc 60 cttcttgctg gcacccaata gaagccatgc gccggaccac gacgtcacgc agcaaaggga 120 cgaggtgtgg gtggtgggca tgggcatcgt catgtctctc atcgtcctgg ccatcgtgtt 180 tggcaatgtg ctggtcatca cagccattgc caagttcgag cgtctgcaga cggtcaccaa 240 ctacttcatc acttcactgg cctgtgctga tctggtcatg ggcctggcag tggtgccctt 300 tggggccgcc catattctta tgaaaatgtg gacttttggc aacttctggt gcgagttttg 360 gacttccatt gatgtgctgt gcgtcacggc cagcattgag accctgtgcg tgatcgcagt 420 ggatcgctac tttgccatta cttcaccttt caagtaccag agcctgctga ccaagaataa 480 ggcccgggtg atcattctga tggtgtggat tgtgtcaggc cttacctcct tcttgcccat 540 tcagatgcac tggtaccggg ccacccacca ggaagccatc aactgctatg ccaatgagac 600 ctgctgtgac ttcttcacga accaagccta tgccattgcc tcttccatcg tgtccttcta 660 cgttcccctg gtgatcatgg tcttcgtcta ctccagggtc tttcaggagg ccaaaaggca 720 gctccagaag attgacaaat ctgagggccg cttccatgtc cagaacctta gccaggtgga 780 gcaggatggg cggacggggc atggactccg cagatcttcc aagttctgct tgaaggagca 840 caaagccctc aagacgttag gcatcatcat gggcactttc accctctgct ggctgccctt 900 cttcatcgtt aacattgtgc atgtgatcca ggataacctc atccgtaagg aagtttacat 960 cctcctaaat tggataggct atgtcaattc tggtttcaat ccccttatct actgccggag 1020 cccagatttc aggattgcct tccaggagct tctgtgtctg cgcaggtctt ctttgaaggc 1080 ctatgggaat ggctactcca gcaacggcaa cacaggggag cagagtggat atcacgtgga 1140 acaggagaaa gaaaataaac tgctgtgtga agacctccca ggcacggaag actttgtggg 1200 ccatcaaggt actgtgccta gcgataacat tgattcacaa gggaggaatt gtagtacaaa 1260 tgactcactg ctgtaacaat tgttgttgtt aacttgttta ttgc 1304 <210> 75 <211> 832 <212> DNA <213> Artificial Sequence <220> <223> Synthetic DNA for plasmid constructs <400> 75 atcttggctg aggaatcttc taacaattta gagcttaaaa acgcccacga ggcggagaac 60 gaaatatcca gagagacgtt agaaacgttc aaaaacgttc gctagcgcca ccatggtgag 120 caagggcgag gagctgttca ccggggtggt gcccatcctg gtcgagctgg acggcgacgt 180 aaacggccac aagttcagcg tgtccggcga gggcgagggc gatgccacct acggcaagct 240 gaccctgaag ttcatctgca ccaccggcaa gctgcccgtg ccctggccca ccctcgtgac 300 caccttcggc tacggcctga tgtgcttcgc ccgctacccc gaccacatga agcagcacga 360 cttcttcaag tccgccatgc ccgaaggcta cgtccaggag cgcaccatct tcttcaagga 420 cgacggcaac tacaagaccc gcgccgaggt gaagttcgag ggcgacaccc tggtgaaccg 480 catcgagctg aagggcatcg acttcaagga ggacggcaac atcctggggc acaagctgga 540 gtacaactac aacagccaca acgtctatat catggccgac aagcagaaga acggcatcaa 600 ggtgaacttc aagatccgcc acaacatcga ggacggcagc gtgcagctcg ccgaccacta 660 ccagcagaac acccccatcg gcgacggccc cgtgctgctg cccgacaacc actacctgag 720 ctaccagtcc aagctgagca aagaccccaa cgagaagcgc gatcacatgg tcctgctgga 780 gttcgtgacc gccgccggga tcactctcgg catggacgag ctgtacaagt ag 832 <210> 76 <211> 227 <212> DNA <213> Artificial Sequence <220> <223> Synthetic DNA for plasmid constructs <400> 76 gaaattaata cgactcacta taggggaccg gtcgccacca tgggctcgag caacctggtt 60 gcgcagctcg aaaacgaagt tgcgtctctg gaaaatgaga acgaaaccct gaagaaaaag 120 aacctgcaca aaaaagacct gatcgcgtac ctggagaaag aaatcgcgaa tctgcgtaag 180 aaaatcgaag aaggcggtgg cgggtcgcaa gagcggcagc agcagct 227 <210> 77 <211> 210 <212> DNA <213> Artificial Sequence <220> <223> Synthetic DNA for plasmid constructs <400> 77 tgcagggtga cactcacgaa ggcggtggcg ggtcgaacct ggttgcgcag ctcgaaaacg 60 aagttgcgtc tctggaaaat gagaacgaaa ccctgaagaa aaagaacctg cacaaaaaag 120 acctgatcgc gtacctggag aaagaaatcg cgaatctgcg taagaaaatc gaagaatgat 180 aatgactcga gaaaatcagc ctcgactgtg 210 <210> 78 <211> 236 <212> DNA <213> Artificial Sequence <220> <223> Synthetic DNA for plasmid constructs <400> 78 gaaattaata cgactcacta taggggaccg gtcgccacca tgggctcgag cgcgcgtaac 60 gcgtatctgc gtaagaaaat cgcacgtctg aaaaaagaca acctgcagct ggaacgtgat 120 gaacagaacc tggaaaaaat catcgcgaac ctgcgtgacg aaatcgcgcg tctcgaaaac 180 gaagttgcgt ctcacgaaca gggcggtggc gggtcgcaag agcggcagca gcagct 236 <210> 79 <211> 219 <212> DNA <213> Artificial Sequence <220> <223> Synthetic DNA for plasmid constructs <400> 79 tgcagggtga cactcacgaa ggcggtggcg ggtcggcgcg taacgcgtat ctgcgtaaga 60 aaatcgcacg tctgaaaaaa gacaacctgc agctggaacg tgatgaacag aacctggaaa 120 aaatcatcgc gaacctgcgt gacgaaatcg cgcgtctcga aaacgaagtt gcgtctcacg 180 aacagtgata atgactcgag aaaatcagcc tcgactgtg 219

Claims

1. A mammalian cell, wherein the cell comprises – A first nucleic acid sequence encoding a first polypeptide fused to the N-terminus of a first variant of a histidine kinase, the histidine kinase comprising a DHp domain and a CA domain. – A second nucleic acid sequence encoding a second polypeptide fused to the N-terminus of a second variant of histidine kinase, the histidine kinase comprising a DHp domain and a CA domain, and – A third nucleic acid sequence that encodes a response regulatory protein that can be specifically phosphorylated by the DHp domain of the first or second variant; – wherein the response regulatory protein comprises a receptor domain fused to an effector domain, wherein the receptor domain is phosphorylated by the DHp domain of the first or second variant, and the effector domain is activated or inhibited by the phosphorylated receptor domain. – The first variant and the second variant are variants of NarX kinase, the receptor domain comprising the NarL response regulatory protein shown in SEQ ID NO: 6, and the effector domain being or comprising the VP16 transcriptional activation domain Vp48 shown in SEQ ID NO: 7; or – wherein the first or second variant comprises the NarX shown in SEQ ID NO: 11 379-598 ; or – Wherein the first variant includes the variant NarX shown in SEQ ID NO: 12 379-598 H399Q, and / or the second variant includes the variant NarX shown in SEQ ID NO:

13. 379-598 N509A.

2. The mammalian cell according to claim 1, wherein it is a human cell.

3. The mammalian cell according to claim 1, wherein – The effector domain is a transcriptional activation domain. – The cell contains a fourth nucleic acid, which contains the target gene controlled by an inducible promoter that can be recognized by the transcriptional activation domain. The activation of the transcriptional activation domain induces the expression of the target gene.

4. The mammalian cell of claim 3, wherein the target gene encodes a target protein or a target RNA.

5. The mammalian cell of claim 4, wherein the target protein is a luminescent protein.

6. The mammalian cell according to claim 3, wherein the inducible promoter is NarL-RE as shown in SEQ ID NO:

2.

7. The mammalian cell of claim 1, wherein – The first variant contains a DHp domain that does not contain histidine residues accessible to the CA domain of the first or second variant of the histidine kinase, and / or – The second variant contains a CA domain that cannot bind to ATP.

8. The mammalian cell of claim 1, wherein the specific binding of the first polypeptide and the second polypeptide is triggerable by a ligand specifically recognized by the first and / or the second polypeptide.

9. The mammalian cell of claim 8, wherein the first polypeptide is a receptor or contains a receptor, the second polypeptide is a binding partner of the receptor or contains a binding partner of the receptor, wherein binding of the receptor and the binding partner can be triggered by the ligand that the receptor can recognize.

10. The mammalian cell of claim 9, wherein the receptor is a transmembrane receptor and the binding partner is a cytoplasmic protein.

11. A method for evaluating protein-protein interactions, wherein the method includes the following steps: – Provides a mammalian cell according to any one of claims 1 to 10, said cell comprising, • The first nucleic acid sequence encodes a first polypeptide fused to the N-terminus of a first variant of histidine kinase, the histidine kinase comprising a DHp domain and a CA domain. • A second nucleic acid sequence encoding a second polypeptide fused to the N-terminus of a second variant of histidine kinase, the histidine kinase comprising a DHp domain and a CA domain, and • A third nucleic acid sequence encoding a response regulatory protein that can be specifically phosphorylated by the DHp domain of the first or second variant, and – Determine the activity of the aforementioned response regulatory protein. in Upon specific binding of the first polypeptide and the second polypeptide, the first and second variants dimerize such that the CA domain of the first or second variant phosphorylates the DHp domain of the first or second variant, and the activity of the response regulatory protein is regulated by phosphorylation of the DHp domain of the first and / or second variant.

12. The method of claim 11, wherein the activity of the response regulatory protein is activated or inhibited by phosphorylation of the DHp domain of the first and / or second variant.

13. A method for evaluating the effect of a compound on protein-protein interactions, wherein the method comprises the following steps: – Provides a mammalian cell according to any one of claims 1 to 10, said cell comprising, • The first nucleic acid sequence encodes a first polypeptide fused to the N-terminus of a first variant of histidine kinase, the histidine kinase comprising a DHp domain and a CA domain. • A second nucleic acid sequence encoding a second polypeptide fused to the N-terminus of a second variant of histidine kinase, the histidine kinase comprising a DHp domain and a CA domain, and • A third nucleic acid sequence encoding a response regulatory protein that can be specifically phosphorylated by the DHp domain of the first or second variant, and – Contact the cells with the compound, and – Determine the activity of the aforementioned response regulatory protein. in Upon specific binding of the first and second peptides, the first and second variants dimerize, such that the CA domain of the first or second variant phosphorylates the DHp domain of the first or second variant, and the activity of the response regulatory protein is modulated by phosphorylation of the DHp domain of the first and / or second variant. The effect of the compound on the specific binding of the first polypeptide and the second polypeptide is determined by the activity of the response regulatory protein.

14. The method of claim 13, wherein the activity of the response regulatory protein is activated or inhibited by phosphorylation of the DHp domain of the first and / or second variant.

15. A method for eliciting a desired response in response to a stimulus, wherein the method comprises the following steps: – Provides a mammalian cell according to any one of claims 1 to 10, said cell comprising, • The first nucleic acid sequence encodes a first polypeptide fused to the N-terminus of a first variant of histidine kinase, the histidine kinase comprising a DHp domain and a CA domain. • A second nucleic acid sequence encoding a second polypeptide fused to the N-terminus of a second variant of histidine kinase, the histidine kinase comprising a DHp domain and a CA domain. The specific binding of the first and second polypeptides is triggered by the stimulant, and • A third nucleic acid sequence encoding a response regulatory protein that can be specifically phosphorylated by the DHp domain of the first or second variant. When the first polypeptide and the second polypeptide specifically bind, the first and second variants dimerize, causing the CA domain of the first or second variant to phosphorylate the DHp domain of the first or second variant, and the activity of the response regulatory protein is regulated by phosphorylation of the DHp domain of the first and / or second variants. – Exposing cells to stimuli, where the required response is mediated by the activity of response regulatory proteins or the activity of response regulatory proteins.

16. The method according to any one of claims 11 to 15, wherein – The response regulatory protein includes a receptor domain fused to an effector domain, wherein the receptor domain is phosphorylated by the DHp domain of the first or second variant, and the effector domain is activated by the phosphorylated receptor domain. – The effector domain is a transcriptional activation domain. – The cell also contains a fourth nucleic acid sequence encoding a target gene, which is under the control of an inducible promoter that can be recognized by the transcriptional activation domain. The activation of the transcriptional activation domain induces the expression of the target gene.

17. The method of claim 16, wherein – The presence of the target gene expression product is determined to be an indicator of the activity of the response regulatory protein, or – The expression product of the target gene is or mediates the desired response.

18. A carrier comprising: – The first nucleic acid sequence as described in any one of claims 1 to 10, – The second nucleic acid sequence as described in any one of claims 1 to 10, and – The third nucleic acid sequence as described in any one of claims 1 to 10.

19. The carrier according to claim 18, further comprising... – The fourth nucleic acid sequence as described in any one of claims 3 to 10.

20. Use of the vector as described in claim 18 or 19 in transfection or transduction of mammalian cells.

21. The use as described in claim 20, wherein the mammalian cell is a human cell.

Images