WO2018069782A2 - A combination of split orthogonal proteases with dimerization domains that allow for assembly - Google Patents
A combination of split orthogonal proteases with dimerization domains that allow for assembly Download PDFInfo
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- WO2018069782A2 WO2018069782A2 PCT/IB2017/055902 IB2017055902W WO2018069782A2 WO 2018069782 A2 WO2018069782 A2 WO 2018069782A2 IB 2017055902 W IB2017055902 W IB 2017055902W WO 2018069782 A2 WO2018069782 A2 WO 2018069782A2
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- proteases
- split
- protease
- orthogonal
- dimerization domains
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
- C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
- C12N9/503—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from viruses
- C12N9/506—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from viruses derived from RNA viruses
Definitions
- the invention relates to a combination of two or more orthogonal split proteases with dimerization domains which allow the assembly of split proteases and to the cells which contain combinations of orthogonal split proteases in combination with target protein.
- Activation of selected cells at a given time and space and the precise control of the cell response to activation with one or more input signals is an important technological problem. Activation can be performed by chemical activators, change in temperature and pH with electrodes or light. Cell activation is also possible with indirect or direct mechanical stimuli such as touch, shear force, fluid flow, hypo- and hyperosmotic stress, and ultrasound.
- synthetic biology often employs artificial signal cascades and logic circuits. Usually, these logical circuits are based on the regulation of transcription and protein translation, which is a relatively slow process, since transcription and translation is required, which means that the response has a delay of more than a few dozen minutes. Faster signaling in cells takes place through posttranslational change of proteins (Olson & Tabor 2012), such as phosphorylation, ubiquitination and proteolytic cleavage.
- proteolysis has already been described as a mechanism for signal transmission, with three known modes of control of protease activity known: autoinhibitor control, proximity sensor and split protease.
- the autoinhibitory peptide is a short amino acid sequence that binds to the active site of the protease, thereby preventing binding of the target substrate. If such autoinhibitory peptide is attached to protease via the cleavage site for the second protease, the activity of the first protease becomes dependent on the activity of the other. For example, a TVMV protease activated by thrombin cleavage was prepared, as well as a HCV protease activated by cleavage with the TVMV protease (Stein & Alexandrov 2014).
- a protease can also be prepared in such a way, so that the binding of the autoinhibitory peptide to the active site is dependent on the presence of the ligand.
- the TVMV protease was prepared, which either activated or deactivated in the presence of a short peptide ligand (Stein & Alexandrov 2014).
- the proximity of the sensor and it's substrate is a way of controlling the activity of the protease, based on the control over the colocation of protease and its substrate.
- cleavage with TEV protease depends on the external signal via the protease linked to the membrane receptor (GPCR), while the luciferase is bound to the receptor (arestin) which binds to the membrane receptor only in the presence of an external ligand, for example a neurotransmitter or hormone, wherein the protease bound to the GPCR triggers the proteolytic splitting of the reporter (Eishingdrelo et al., 2011).
- the autoinhibitory proteases with HCV and TVMV described above were enclosed in a proximity sensor by binding them to the FKBP and FRB domains (Stein & Alexandrov 2014). These domains are linked in the presence of rapamycin, which also brings the proteases to close proximity. If one of the proteases in the binding peptide between the enzyme and the autoinhibitory peptide contains a target cleavage site for the second protease, their immediate proximity allows the autoinhibitory peptide to be disconnected and thereby the system is activated.
- the third method of induction of protease with ligand, split protease has been described so far only for the TEV protease (Wehr et al., 2006).
- the protease sequence was split into the N-terminal and C- terminal fragments attached to the rapamycin-binding domains of FKBP and FRB. In the presence of rapamycin, these domains connect and being both halves of split proteases in close proximity which allows the protease to take up its active form.
- Each of the coiled coils contains a cleavage site fused with an autoinhibitory peptide which inhibits coiled coil dimerization. After both autoinhibitory peptides have been cleavage by a protease (TEV and caspase-3 have been used) dimerization is enabled and an output signal is detected.
- the described reporter acts as a logic operation AND.
- Protease TEV belongs to the family of potivirus proteases. These viruses are plant pathogens whose genome is transcribed into a single polyprotein, a part of which is a core inclusion domain (NIa) that splits the viral polyprotein into individual functional subunits.
- TEV protease In addition to the TEV protease, PPV, TVMV, SbMV, SuMMV and other protease viruses are known and characterized in this family, and target sequences are also known which are effectively cleaved by these proteases (JM Adams et al., 2005; MJ Adams et al. 2005; Ghabrial et al., 1990; Tozser et al., 2005). Some of these proteases have already been shown to be orthogonal (Fernandez-Rodriguez & Voigt 2016). Nevertheless, only the TEV protease appears as a widely used synthesis-biological tool and is the only potivirus protease that has been prepared so far in split form.
- RAFT1 a mammalian protein that binds to FKBP12 in a rapamycin-dependent fashion and is homologous to yeast TORs. Cell, 78(1), pp.35-43. Sabers, CJ. et al., 1995. Isolation of a protein target of the FKBP12-rapamycin complex in mammalian cells. The Journal of biological chemistry, 270(2), pp.815-822.
- the invention solves the above-described problems of lack of fast orthogonal signaling pathways based on posttranslational modification of proteins, which was achieved by the introduction of a combination of at least two orthogonal split proteases in association with dimerization domains that allow the reconstitution of split proteases into cells.
- the invention relates to a combination of orthogonal split proteases that recognize a target sequence comprising at least 6 amino acids associated with dimerization domains that allow for the reconstitution of split proteases.
- At least two orthogonal proteases are prepared as split fragments in association with dimerization domains, where dimerization can be inducible by light, chemical or other input signal.
- Proteases cleave one or more target proteins that contain a target sequence for one or more orthogonal proteases and act as further mediators of the signal, the reporting protein, or therapeutic proteins. With appropriately selected target proteins, logical circuits mediated by proteases can be prepared.
- the invention also relates to cells that contain a combination of orthogonal split proteases in association with dimerization domains and target proteins containing the appropriate cleavage sites identified by individual proteases when they are assembled.
- a complementary pair of dimerization domains allows for the assembly of individual split proteases.
- the complementary pair can be formed spontaneously or with the help of an initiator, which can be intracellular or external, such as light, pH, temperature, mechanical stress, chemical signal.
- Dimerization domains can be different for each type of split protease or are the same for all orthogonal proteases.
- Reconstituted protease acts on a target protein containing the cleavage site for the selected protease which may be endogenous or transgenetically inserted into a reporter or other intermediary signal.
- split proteases After addition of the ligand or another signal (e.g., light) which causes the association of the dimerization domains, structural and functional reconstitution of the split proteases is achieved, which cleave the next member of the signaling cascade containing the target peptide sequence for cleavage with the selected protease.
- a split protein may be a direct reporter (for example, luciferase or a fluorescence protein) or the next mediator of the signal cascade (for example, a transcription factor, kinase, phosphatase, or another protease), and its main feature is that its activity depends on cleavage.
- a signaling system based on split proteases contains at least two split proteases.
- Each of the split proteases in the system may have its target protein (mediator or reporter), but two or more proteases may function on the same target protein if it contains various identifiable cleavage sites.
- the concept of the invention is presented graphically in Figure 1.
- Transmission of the signal through split proteases may trigger or stop the transcription of selected genes or secretion of selected molecules, which is useful for the treatment of nervous system disorders, hormonal disorders, for metabolic and blood flow restrictions and other diseases.
- the invention is useful for controlling the action of human, animal or plant cells, for transmitting the signal to other cells, for secreting peptides, proteins and other molecules, to detect a combination of two or more chemical, light or mechanical signals.
- Figure 1 A schematic diagram of a signal system with orthogonal split proteases associated with dimerization domains that can be activated via an inductor that may be intracellular or external.
- the inactive split protease is assembled into the active protease and cleaves the target protein substrate, which leads to a change in the target protein, which may be a reporter, a different protease, an inactive enzyme, etc.
- Each orthogonal split protease can have its own dimerization domains, thus various inductors can activate various orthogonal proteases, and in this way lead to different cell responses via logic operations,.
- Proteases break down the target protein that leads to the continuation of the signal pathway (for example, transcriptional regulation) or represents an output signal (for example a fluorescent protein). If the target protein contains recognizable sites for cleavage with various proteases, the signal transfer with proteases can act as a logic circuit.
- Figure 2 Orthogonality of selected proteases. The activity of each protease is shown as a cyclic luciferase activity with a marked cleavage site for the protease. The target sequences for cleavage of each protease are shown.
- Figure 3 Inducibility of selected proteases. The activity of each protease is shown as cyclic luciferase activity with the appropriate cleavage site for the selected protease. A) Induction of split proteases with rapamycin. B) Induction of the split TEV protease with blue light. C) Induction of split PPV protease with blue light.
- FIG. 4 Activation of orthogonal signaling pathways mediated by TEV and PPV proteases.
- TEV protease cleaves the transcription factor, which increases the expression of the mCitrin reporter.
- PPV protease activates the cyclic luciferase reporter via cleavage.
- Figure 5 The NOR logical operation mediated by TEV and PPV proteases.
- FIG. 6 A of NIMPLY B logic function mediated by TEV split protease with light induction and PPV split protease by chemical induction.
- the activity of the reporter is high only when stimulated with light that activates the TEV protease, but not with stimulation with rapamycin that activates PPV protease or with rapamycin and light at the same time.
- the invention relates to a combination of two or more orthogonal split proteases that recognize a target sequence comprising of at least 6 amino acids with dimerization domains that enable the functional reconstitution of protease activity, as well as target proteins with a target sequence recognized by the proteases.
- the combination of orthogonal split proteases with dimerization domains includes, according to the invention, :
- At least two split proteases each of which is expressed as a minimal of two split protein fragments and which are selected preferentially from the NIa family of potivirus proteases, preferentially proteins SuMMV, SbMV, PPV and TEV; preferentially at least one of the split proteases is composed of two proteins with the protein sequence of SEQ ID 4 and SEQ ID 6, SEQ ID. 10 and SEQ ID. 12, SEQ ID. 16 and SEQ ID. 18, SEQ ID. 22 and SEQ ID. 24, or their homologs that have at least 30% similarity of the amino acid sequence
- dimerization domains that assemble spontaneously and are selected from domains that spontaneously form dimers, preferentially dimeric proteins, helical coils and beta structures, all of which have, preferentially, orthogonal properties
- the dimerization of the domains is triggered by (i) an intracellular signal such as: a change in the concentration of a metabolite or a secondary signaling molecule, a change in the enzyme activity, or with (ii) an external signal such as temperature change, pH value, mechanical stress, change in osmotic pressure, ultrasound, light, chemical or biological signal
- dimerization domains preferentially coiled helices, light inducible domains; preferentially CRY2 and CIBN, chemically inducible domains; preferentially FKBP and FRB, or calcium-dependent domains; preferably calmodulin and Ml 3, in fusion with split protease fragments that can form homodimers, heterodimers or which form a covalent bond as a result of self cleavage (inteins) and whose dimerization is dependent on the input signal, the input signal being a mechanical stimulus; preferably ultrasound or touch, a light signal, a chemical ligand; preferably rapamycin, or another physiologically relevant signal, such as proteolytic cleavage
- At least one protein comprising at least one recognizable cleavage site for at least one of the split proteases and may form the next sequential step in a signaling pathway, for instance a transcriptional regulator, or which functions as a therapeutic target, a therapeutic protein or peptide or a reporter protein, preferably luciferase, SEAP or a fluorescent protein
- Optionally includes an arbitrary marker sequence for protein detection or affinity chromatography (e.g., HIS and AU1 markers).
- an arbitrary marker sequence for protein detection or affinity chromatography e.g., HIS and AU1 markers.
- the invention relates to cells that contain the above-described orthogonal signaling pathways and in which the signal path leads to a response that can be expressed in several ways, such as (i) release of endogenous cell metabolites, peptides or proteins (e.g., hormones), ( ii) releasing exogenous metabolites, peptides or proteins, preferably therapeutic proteins, (iii) modifying the activity of endogenous or exogenous proteins (preferably enzymes) in the cell, or (iv) controlling the expression of genes.
- the invention relates to cells selected from bacterial or eukaryotic cells, plant cells or human cell lines, preferentially the invention relates to mammalian cells and human cell lines, for example to neurons or other nervous system cells, T lymphocytes or other immune cells or pancreatic beta cell.
- split protease refers to two or more polypeptides ("fragments") derived from the protease sequence, each of them being equal to one part of the whole protease.
- fragments alone are not catalyticaly active.
- the formation of a proteolytically active complex requires the interaction of protease fragments; this process is termed "reconstitution”.
- Protease fragments are selected so that reconstitution cannot occur between them without the aid of additional dimerization domains, which are expressed in fusion with protease fragments in the form of a chimeric protein.
- chimeric protein means a protein or polypeptide consisting of sequences derived from non-native proteins and formed upon the translation of a chimeric nucleic acid that combines the records for the individual domains of non-native proteins composed to form a single open reading frame.
- dimerization domain refers to protein domains that connect independently or in the presence of a ligand to one another through covalent or non-covalent interactions.
- homodimerization refers to domains that connect to other domains of the same type, and the term “heterodimerization” refers to domains that connect to other domains of a different type.
- constitutive dimerization domains refers to dimerization domains that connect themselves independently, such as, for example, coiled coils, and the term “inducible dimerization domain” refers to dimerization domains that merge only in the presence of a dimerization signal.
- signal refers to a measurable change within a cell or in its environment.
- input signal refers to a change that triggers a signalling pathway
- output signal refers to a change that occurs as a result of the signalling pathway.
- An input signal may be part of an endogenous signalling pathway or is triggered exogenously and causes a physiological response of the cell, for example, a change in the concentration of a secondary signalling molecule, such as calcium, or a dimerization signal that affects the dimerization of endogenous or exogenous dimerization domains.
- dimerization signal refers to ligands, light and mechanical stimuli, pH changes, post- translational modification of dimerization domains, change in the secondary signalling molecule concentration, or alteration of the transmembrane potential, which cause the dimerization of domains that do not have intrinsic affinity with each other and cannot be connected independently in the absence of a dimerization ligand or signal.
- mechanical signal refers to all kinds of mechanical disturbances / forces such as ultrasound, touch, osmotic stress and friction due to the flow of fluid that affect the cells or the cell membrane.
- the mechanical signal can be generated by the pressure of a particular solid object, liquid, or other cells on the cell membranes, due to the gravitational, centrifugal, shear or other direct force, or due to indirect forces that induce action on the cell membrane, such as osmosis, stretching or shrinking of the membrane due to temperature or other factors.
- ultrasonic refers to acoustic waveforms with a frequency between about 20 kHz to about 15 MHz, generated by a device with which the strength or sound amplitude, frequency in combination with a suitable converter, and a time dependency regime for ultrasonic pulses can be controlled.
- the term »chemical signal « used herein refers to a change in ligand concentration.
- the term »ligand « used herein refers to short biopolymers, organic or inorganic molecules and ions that can bind to proteins, preferably to dimerization domains.
- light signal refers to electromagnetic waves in the visible range (wavelengths between 400 and 700 nm), near infrared (wavelengths between 700 nm and 4 ⁇ ) or ultraviolet (wavelengths between 10 nm and 400 nm), which may cause conformational or chemical changes in biological molecules, preferably dimerization domains or their ligands.
- signal pathway refers to one or more events in a cell that may comprise ligand binding, dimerization of proteins or peptides, influx of secondary messenger molecules, alteration of the transmembrane potential, enzyme-catalyzed reaction or other cell changes, and which are triggered by an input signal, and which produces an output signal as its final product.
- orthogonal proteases means two or more proteases that differ in the order of the target substrate so that none of the orthogonal proteases do not split the target substrate of the second orthogonal protease.
- inducible protease refers to the property of the protease, by which its enzymatic activity significantly changes in the presence of an input signal.
- the input signal can also be the output signal of a separate signalling pathway.
- linker peptide refers to amino acid sequences whose role is to separate the individual domains of the assembled protein and to enable their proper spatial orientation.
- the role of the linker peptide in the fusion protein can also be used as a space to include the insertion of the cleavage site, the posttranslational modification site, the marker sequence, or the localization signal.
- the term »cleavage site refers to the amino acid sequence to which a specific protease can bind and within which the protein is cleaved by hydrolysis of the peptide bond.
- the term »marker sequences refers to the amino acid sequences added to the protein for easier purification, isolation, or detection of the protein.
- the term localization signal refers to an amino acid sequence that directs the protein to a particular location in the cell. Localization signals vary according to the host organism in which the protein is expressed. The amino acid sequences of localization signals are well known among experts in the field and it is also known which signal sequence operates in specific organisms.
- linker peptides The position of linker peptides, marker sequences, cleavage sites, and localization signals is arbitrary, although they must be posited in a way that allows for the functional expression of the protein and preserves the functions for which these amino acid sequences have been selected and which are well known to experts in the field.
- cell refers to an eukaryotic cell, a cellular or multicellular organism (cell line) cultured as a single cell entity that has been used as a recipient of nucleic acids and includes the daughter cells of the original cell that has been genetically modified by the inclusion of nucleic acids.
- the term refers primarily to cells of higher developed eukaryotic organisms, preferably vertebrates, preferably mammals.
- Genetically modified host cell (also “recombinant host cell”) is a host cell into which the nucleic acid has been introduced.
- the eukaryotic genetically modified host cell is formed in such a way that a suitable nucleic acid or recombinant nucleic acid is introduced into the appropriate eukaryotic host cell.
- the invention hereafter includes host cells and organisms that contain a nucleic acid according to the invention (transient or stable) bearing the operon record according to the invention.
- Suitable host cells are known in the field and include eukaryotic cells. It is known that proteins can be expressed in cells of the following organisms: human, rodent, cattle, pork, poultry, rabbits and the like. Host cells may include cultured cell lines of primary or immortalized cell lines.
- nucleic acids refers to a polymeric form of nucleotides (ribonucleotides or deoxyribonucleotides) of any length and is not limited to single, double or higher chains of DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids, or polymers with a phosphorothioate polymer backbone made from purine and pyrimidine bases or other natural, chemical or biochemically modified, synthetic or derived nucleotide bases.
- »polypeptide«, »protein « , »peptide «, used herein refers to the polymeric forms of amino acids of any length.
- «functional polypeptide* used herein refers to a polypeptide form of any length of amino acids which expresses any function, for instance: structure formation, localizing to a specific location, localizing to specific organelles, facilitating and triggering chemical reactions, binding to other functional polypeptides.
- heterologous refers to the context of genetically modified host cells and refers to a polypeptide for which at least one of the following claims applies: (a) the polypeptide is foreign ("exogenous") to the host cell (it is not naturally present within the cell); (b) the polypeptide is naturally present ("endogenous") in a given host microorganism or host cell, but is produced in an unnatural amount in the cell (more than expected or in greater amounts than found in nature) or is differentiated in the nucleotide sequence from the endogenous nucleotide sequence, so that the same protein (having the same or substantially similar amino acid sequence) as the endogenous protein is produced in unnatural amounts (more than expected or more than found in nature) in the cell.
- homologous refers to proteins or nucleic acids with a well-conserved amino acid or nucleotide sequences, preferably with at least 50% conservation and no less than 20% conservation, determined by protein or nucleic acid comparative techniques known to experts in the field.
- homologous nucleic acids encode homologous proteins.
- a particular nucleic acid (DNA or RNA) is a product of various combinations of cloning, restriction and / or ligation leading to a construct having structurally coding or non-coding sequences different from endogenous nucleic acids in a natural host system.
- a DNA sequence encoding a structurally coding sequence may be formed from cDNA fragments, from short oligonucleotide linkers or from synthetic oligonucleotides from which a synthetic nucleic acid is obtained which can be expressed on a recombinant transcription unit in a cellular or cell- free transcription and translation system.
- Such a sequence can be used in the form of an open reading frame in which there is no interference from the internal non-translated sequences (introns) which are usually present in eukaryotic genes.
- Genomic DNA which contains important sequences can also be used to form a recombinant gene or transcription unit.
- Sequences of untranslated DNA may be present at the 5'- or 3'-end of the open reading frame, where such sequences do not affect the manipulation or expression of the coding regions and can act as modulators for the production of desired products through various mechanisms.
- the insertion of the vectors into the host cells is carried out by conventional methods known from the field of science, and the methods relate to transformation or transfection and include: chemically induced insertion, electroporation, micro-injection, DNA lipofection, cellular sonication, gene bombardment, viral DNA input, as well as other methods.
- the entry of DNA may be of transient or stable.
- Transient refers to the insertion of a DNA with a vector that does not incorporate the DNA of the invention into the cell genome.
- a stable insertion is achieved by incorporating DNA of the invention into the host genome.
- the insertion of the DNA of the invention in particular for the preparation of a host organism having stably incorporated DNA of the invention, can be screened by the presence of markers.
- the DNA sequence for markers refers to resistance to antibiotics or chemicals and may be included on a DNA vector of the invention or on a separate vector.
- Example 1 Preparation of cells, which feature a signaling pathway mediated by split proteases
- DNA constructs Preparation of DNA records for orthogonal split proteases and their targets.
- the inventors used molecular biology methods such as: chemical transformation of competent E. coli cells, plasmid DNA isolation, polymerase chain reaction (PCR), reverse transcription - PCR, PCR linking, nucleic acid concentration determination, DNA agarose gel electrophoresis, isolation of fragments of DNA from agarose gels, chemical synthesis of DNA, DNA restriction with restriction enzymes, cutting of plasmid vectors, ligation of DNA fragments, purification of plasmid DNA in large quantities.
- PCR polymerase chain reaction
- PCR reverse transcription -PCR
- PCR linking nucleic acid concentration determination
- DNA agarose gel electrophoresis isolation of fragments of DNA from agarose gels
- chemical synthesis of DNA DNA restriction with restriction enzymes
- cutting of plasmid vectors ligation of DNA fragments
- purification of plasmid DNA purification of plasmid DNA in large quantities.
- the proteases were selected from the proteases of the NIa family of potivirus proteins, specifically the SuMMV, SbMV, PPV and TEV proteases. Cleavage sequences for each of the described proteases are known to experts in the field. Based on the amino acid sequence similarity between these proteases and the already known sequence of split TEV protease, we prepared nucleotide transcripts for split proteases in fusion with dimerization domains.
- the light-induced domains of CRY2 and CIBN (Kennedy et al., 2010) and the rapamycin inducible domains of FKBP and FRB were selected as dimerization domains (Sabatini et al., 1994; Brown et al., 1994; Sabers et al., 1995).
- Table 1 List of sequences of split proteases that were used to illustrate the invention and their recognizable cleavage sites
- Table 2 Split proteases with dimerization domains that were used to illustrate the invention.
- Table 3 Fusion proteins and operones which were used, appart from the split proteases, to illustrate the invention.
- Cells from the HEK293T cell line were grown at 37 ° C and 5% C02.
- a DMEM medium containing 10% FBS was used, containing all the necessary nutrients and growth factors.
- the cells were grafted into a new breeding flask and / or diluted.
- the number of cells was determined by a hemocytometer and seeded in density 2.5xl0 4 per hole into the microtiter plate with 96 holes 18-24 hours before transfection.
- the seeded plates were incubated at 37 ° C and 5% C02 until the cells were 50-70% confluent and ready for transfection by transfection reagent.
- the transfection was carried out according to the instructions of the transfection reagent manufacturer (e.g., JetPei, Lipofectamine 2000) and was adapted for the microtiter plate used.
- Example 2 Catalytic activity of split proteases in mammalian cells at stimulation with various signals and orthogonality of cleavage with individual protease Cyclic firefly luciferase Flue based reporter system (Kanno et al. 2007) was used to detect catalytic activity of examined proteases. Cyclic luciferase, used as a reporter protein, was prepared by reorganizing amino acid sequence of firefly luciferase. Amino acids 4-233 from N-terminus were placed downstream to the C-terminus, connecting the two domains by a short linker which contains cleavage site for one of the following proteases: TEV, PPV, SuMMV or SbMV.
- the newly prepared protein was further modified by fusing two fragments of an intein via short linker to the ends, namely C-fragment of intein DnaE from organism Nostoc punctiforme was fused to N-terminus and N-fragment of intein DnaE from organism N. punctiforme to the C-terminus followed by amino acid sequence of peptide PEST-CL1 for fast digestion of the protein, previously known to experts from this field of science.
- Prepared luciferase based reporter thus forms inactive cyclic proteins, which can be cleaved with a target protease, resulting in structural reorganization and gain of luciferase catalytic activity.
- HEK293T cells implanted in 96-well plates, were transfected one day prior to the experiment with plasmids, which encode for one of examined proteases or two proteins, that combined form an active split protease; plasmids that encode previously described cyclic luciferase as a reporter protein and plasmid that encodes for luciferase from organism Renilla reniformis Rluc (GenBank AF362545.1).
- Cells were stimulated by addition of rapamycin to end concentration of 1 ⁇ one day prior to measurement or with light beam (455 nm wavelength) in specially prepared machine 30min prior to measurement.
- protease-cleavage- dependentTAL (N) -VP16-TEVs-KRAB transcription factor in addition to cyclic luciferase.
- the transcription factor was prepared in such a way that the sequence for the VP 16 activation domain was aded at the C-terminus of the TAL DNA binding domain, which is well known to the field experts, followed by a binding peptide with a recognizable sequence for cleavage with the TEV protease and the repressive domain KRAB.
- the VP16 and KRAB domains are also well-known to the field experts.
- the transcription factor changes the mode of action from repression to activation of the transcription of genes.
- a gene for the yellow fluorescence protein mCitrine was inserted into the plasmid after a minimal promoter with binding sites for the DNA-binding TAL domain.
- the HEK293T cells implanted in 96-hole plates, were transfected one day before the experiment with plasmids bearing cyclic luciferase code with a recognizable cleavage site with PPV protease, a transcription factor TAL (N) -VP16-TEVs-KRAB, mCitrin under the appropriate promoter and a code for luciferase Rluc, as well as plasmid with a TEV or PPV protease code as marked.
- TAL transcription factor TAL
- the media was removed from the cells and then measured by a fluorescence reader of SynergyMX plates (manufactured by BioTek) with excitation at the wavelength of 512 nm and an emission at the wavelength of 532 nm. The fluorescence of untransfected cells was subtracted from the value obtained.
- the reaction cyclic luciferaseprotein activity the cells were lysed with buffer according to the manufacturer's instructions (Promega) and then the activity of FLuc and Rluc was measured as in the above experiment.
- Logical functions NOR and A NIMPLY B have been developed as examples of the logical circuit with the transmission of the signal with proteases, the latter being the equivalent composite logic function A AND NOT B.
- a split luciferase rapporteur was used, which is comprised of an N-terminal luciferase fragment linked via a cleavage site for the TEV protease with an AP4 peptide and a C -terminal luciferase fragment, which is coupled via a cleavage site for the PPV protease with the peptide P3.
- Peptides P3 and AP4 form a coiled coil, which allows for the reconstitution of luciferase fragments into an active enzyme.
- the HEK293T cells in the 96-hole plate were transfected one day before the experiment with plasmids bearing the Rluc luciferase, the nLuc-TEVs-AP4 and P3-PPVs-cLuc luciferase, and plasmids with TEV protease and PPV protease as marked.
- the activity of the reporting proteins Flue and Rluc was measured as described above. Result:From Fig. 5B it can be seen that we observed a decrease in the activity of the reporter when coexpressed with any of the selected proteases. This is in line with the expected function of the NOR function.For the function A NIMPLY B (Fig.
- a split protease reporter was also used, but in this case it consists of an N-terminal luciferase associated with the peptide AP4, which is further linked to the P4mS peptide by the cleavage site for the TEV protease.
- a C-terminal luciferase fragment bound to the P3 peptide via the cleavage site for the PPV protease was added to the system as well.
- the peptides AP4 and P3mS form a coiled coil, which prevents the formation of a coiled coil between AP4 and P3 and thus the reconstitution of the functional luciferase enzyme.
- the formation of a coiled coil between AP4 and P3mS destabilizes as it cleaves the connecting peptide between them. Since the peptide P3 forms a more stable coiled coil with AP4, now there may be reconstitution of luciferase, but only in the absence of PPV protease, since it cleaves the C- terminal luciferase fragment from P3 peptide. The outcome signal (activity of luciferase) can therefore be detected only when cleavage with TEV protease and absence of cleavage with PPV protease.
- the HEK293T cells in the 96-hole plates were transfected one day before the experiment with plasmids bearing the Rluc luciferase, the nLuc-AP4-TEVs-P3mS and P3-PPVs-cLuc reporting protein, plasmids with split TEV with inducible CRY2 and CIBN domains, and plasmids bearing splice-protected PPV proteases with inducible FKBP and FRB domains.
- the cells were stimulated by the addition of rapamycin to a final concentration of 1 ⁇ or with light with a wavelength of 455 nm 15 minutes before the measurement in a specially prepared device. The activity of the reporting Flue and Rluc protein was measured as described above.
Abstract
Invention refers to combination of split orthogonal proteases that recognize and cleave target sequence of at least 6 amino acids. Parts of split orthogonal proteases are fused to dimerization domains that allow formation of the whole protease from two split protease parts. At least two orthogonal proteases are designed as split fragments fused to dimerization domains where dimerization can be induced with either light, chemicals or other input signal. Proteases cleave one or more target proteins that include cleavage site for one or more orthogonal proteases and act as a signal transducers, reporters or therapeutic proteins. With appropriately selected target proteins, logical circuits mediated by proteases can be prepared. The invention also relates to cells that contain expressed split proteases to transmit the signal.
Description
A combination of split orthogonal proteases with dimerization domains that allow for assembly
Field of invention
The invention relates to a combination of two or more orthogonal split proteases with dimerization domains which allow the assembly of split proteases and to the cells which contain combinations of orthogonal split proteases in combination with target protein.
State of the art
Activation of selected cells at a given time and space and the precise control of the cell response to activation with one or more input signals is an important technological problem. Activation can be performed by chemical activators, change in temperature and pH with electrodes or light. Cell activation is also possible with indirect or direct mechanical stimuli such as touch, shear force, fluid flow, hypo- and hyperosmotic stress, and ultrasound. To control the cell response to activation, synthetic biology often employs artificial signal cascades and logic circuits. Usually, these logical circuits are based on the regulation of transcription and protein translation, which is a relatively slow process, since transcription and translation is required, which means that the response has a delay of more than a few dozen minutes. Faster signaling in cells takes place through posttranslational change of proteins (Olson & Tabor 2012), such as phosphorylation, ubiquitination and proteolytic cleavage.
So far, proteolysis has already been described as a mechanism for signal transmission, with three known modes of control of protease activity known: autoinhibitor control, proximity sensor and split protease.
The autoinhibitory peptide is a short amino acid sequence that binds to the active site of the protease, thereby preventing binding of the target substrate. If such autoinhibitory peptide is attached to protease via the cleavage site for the second protease, the activity of the first protease becomes dependent on the activity of the other. For example, a TVMV protease activated by thrombin cleavage was prepared, as well as a HCV protease activated by cleavage with the TVMV protease (Stein & Alexandrov 2014). With a suitable selection of linking peptides and inducible dimerization domains, a protease can also be prepared in such a way, so that the binding of the autoinhibitory peptide to the active site is dependent on
the presence of the ligand. Thus, the TVMV protease was prepared, which either activated or deactivated in the presence of a short peptide ligand (Stein & Alexandrov 2014).
On the other hand, the proximity of the sensor and it's substrate is a way of controlling the activity of the protease, based on the control over the colocation of protease and its substrate. For example, cleavage with TEV protease depends on the external signal via the protease linked to the membrane receptor (GPCR), while the luciferase is bound to the receptor (arestin) which binds to the membrane receptor only in the presence of an external ligand, for example a neurotransmitter or hormone, wherein the protease bound to the GPCR triggers the proteolytic splitting of the reporter (Eishingdrelo et al., 2011). Similarly, the autoinhibitory proteases with HCV and TVMV described above were enclosed in a proximity sensor by binding them to the FKBP and FRB domains (Stein & Alexandrov 2014). These domains are linked in the presence of rapamycin, which also brings the proteases to close proximity. If one of the proteases in the binding peptide between the enzyme and the autoinhibitory peptide contains a target cleavage site for the second protease, their immediate proximity allows the autoinhibitory peptide to be disconnected and thereby the system is activated.
The third method of induction of protease with ligand, split protease, has been described so far only for the TEV protease (Wehr et al., 2006). The protease sequence was split into the N-terminal and C- terminal fragments attached to the rapamycin-binding domains of FKBP and FRB. In the presence of rapamycin, these domains connect and being both halves of split proteases in close proximity which allows the protease to take up its active form.
With an appropriate arrangement of the target cleavage sites on the protease reporter or other intermediate proteins we may also monitor how the activity of several different proteases reflects differently on the reporter. So far, control over genetic circuts using orthogonal proteases which cleavage transcription factor inhibitory domains or degron fused on a transcription factor which exposes the transcription factor to degradation, have been reported (Fernandez-Rodriguez & Voigt 2016). Shekhawat and colleagues (Shekhawat et al. 2009) have developed a reporter composed of two fragments of split luciferase, each of them coupled to a coiled helix and dimerization is enabled by coiled coil pairing. Each of the coiled coils contains a cleavage site fused with an autoinhibitory peptide which inhibits coiled coil dimerization. After both autoinhibitory peptides have been cleavage by a protease (TEV and caspase-3 have been used) dimerization is enabled and an output signal is detected. The described reporter acts as a logic operation AND.
Protease TEV belongs to the family of potivirus proteases. These viruses are plant pathogens whose genome is transcribed into a single polyprotein, a part of which is a core inclusion domain (NIa) that splits the viral polyprotein into individual functional subunits. In addition to the TEV protease, PPV, TVMV, SbMV, SuMMV and other protease viruses are known and characterized in this family, and target sequences are also known which are effectively cleaved by these proteases (JM Adams et al., 2005; MJ Adams et al. 2005; Ghabrial et al., 1990; Tozser et al., 2005). Some of these proteases have already been shown to be orthogonal (Fernandez-Rodriguez & Voigt 2016). Nevertheless, only the TEV protease appears as a widely used synthesis-biological tool and is the only potivirus protease that has been prepared so far in split form.
The literature
Adams, J.M., Antoniw, F.J. & Fauquet, M.C., 2005. Molecular criteria for genus and species discrimination within the family Potyviridae. Archives of Virology, 150(3), pp.459-479.
Adams, M.J., Antoniw, J.F. & Beaudoin, F., 2005. Overview and analysis of the polyprotein cleavage sites in the family Potyviridae. Molecular Plant Pathology, 6(4), pp.471-487.
Brown, E.J. et al., 1994. A mammalian protein targeted by Gl-arresting rapamycin-receptor complex. Nature, 369(6483), pp.756-758.
Eishingdrelo, H. et al., 2011. A cell-based protein-protein interaction method using a permuted luciferase reporter. Current chemical genomics, 5, pp.122-128.
Fernandez-Rodriguez, J. & Voigt, C.A., 2016. Post-translational control of genetic circuits using Potyvirus proteases. Nucleic acids research, 44(13), pp.6493-502.
Ghabrial, S.A. et al., 1990. Molecular genetic analyses of the soybean mosaic virus NIa proteinase. The Journal of general virology, 71 (9), pp.1921-7.
Kanno, A. et al., 2007. Cyclic luciferase for real-time sensing of caspase-3 activities in living mammals. Angewandte Chemie (International ed. in English), 46(40), pp.7595-7599.
Kennedy, M.J. et al., 2010. Rapid blue-light-mediated induction of protein interactions in living cells. Nature methods, 7(12), pp.973-975.
Olson, E.J. & Tabor, J.J., 2012. Post-translational tools expand the scope of synthetic biology. Current opinion in chemical biology, 16(3-4), pp.300-6.
Sabatini, D.M. et al., 1994. RAFT1 : a mammalian protein that binds to FKBP12 in a rapamycin-dependent fashion and is homologous to yeast TORs. Cell, 78(1), pp.35-43.
Sabers, CJ. et al., 1995. Isolation of a protein target of the FKBP12-rapamycin complex in mammalian cells. The Journal of biological chemistry, 270(2), pp.815-822.
Shekhawat, S.S. et al., 2009. An Autoinhibited Coiled-Coil Design Strategy for Split-Protein Protease Sensors. Jacs, 131(42), pp.15284-15290.
Stein, V. & Alexandrov, K., 2014. Protease-based synthetic sensing and signal amplification. Proceedings of the National Academy of Sciences of the United States of America, 111(45), pp.15934-9.
Tozser, J. et al., 2005. Comparison of the substrate specificity of two poty virus proteases. FEBS Journal, 272(2), pp.514-523.
Wehr, M.C. et al., 2006. Monitoring regulated protein-protein interactions using split TEV. Nature methods, 3(12), pp.985-993.
The technical problem
For precise management and selectivity of biological systems, it is desirable to combine several signals in order to test different combinations of physiological conditions, cells or signals from the environment. Natural cells do this via signal paths. The problem is to design a new orthogonal signal path, meaning only a selected cell compartment will be affected in a specific way.
For the design of new orthogonal signal paths within human cells, we found that signaling through proteolysis represents an opportunity for the construction of a system for rapid signal transmission that has not yet been utilized. In order to operate within cells, it is best to select proteases that operate on longer target sequences containing at least 6 amino acids, which makes it unlikely that they can impair the proteins that are needed for the cell functioning. On the other hand, in this way we can insert specific cleavage sites into selected proteins for cleavages with specific protease or their combinations.
A significant lack of orthogonal proteases, which act on different, well-defined longer polypeptide sequences and whose activity could be directly dependent on the input signal, was observed in this.
For the purposes of more complex control of the cell response to various external signals, we need a larger number of signal molecules and cascades that interact either orthogonally or complement to each other and act as logical circuits. In particular, it is important that the activity of the transferring molecules in the signal cascade (a protease in the above mentioned example) is precisely controlled and orthogonal. The selected proteases must therefore: a) be inactive in the absence of a signal, b) allow easy reconstitution of the activity in the presence of the signal, c) not affect the endogenous processes in the
cell, and d) be orthogonal, therefore the transmission of the signal over one part of the cascade does not affect the transmission of the signal over the other part of the cascade, except when intentionally planned.
It is evident from the state of the art that current solutions cannot provide accurate, fast, logically- based and selective management of biological systems at several levels coupled with different or identical input signals.
The problem solution
The invention solves the above-described problems of lack of fast orthogonal signaling pathways based on posttranslational modification of proteins, which was achieved by the introduction of a combination of at least two orthogonal split proteases in association with dimerization domains that allow the reconstitution of split proteases into cells.
The invention relates to a combination of orthogonal split proteases that recognize a target sequence comprising at least 6 amino acids associated with dimerization domains that allow for the reconstitution of split proteases. At least two orthogonal proteases are prepared as split fragments in association with dimerization domains, where dimerization can be inducible by light, chemical or other input signal. Proteases cleave one or more target proteins that contain a target sequence for one or more orthogonal proteases and act as further mediators of the signal, the reporting protein, or therapeutic proteins. With appropriately selected target proteins, logical circuits mediated by proteases can be prepared.
The invention also relates to cells that contain a combination of orthogonal split proteases in association with dimerization domains and target proteins containing the appropriate cleavage sites identified by individual proteases when they are assembled. A complementary pair of dimerization domains allows for the assembly of individual split proteases. The complementary pair can be formed spontaneously or with the help of an initiator, which can be intracellular or external, such as light, pH, temperature, mechanical stress, chemical signal. Dimerization domains can be different for each type of split protease or are the same for all orthogonal proteases.
Reconstituted protease acts on a target protein containing the cleavage site for the selected protease which may be endogenous or transgenetically inserted into a reporter or other intermediary signal.
We selected a family of potivirus proteases, of which TEV protease is also a member, and invented new split proteases based on the sequential and functional similarities between the individual
proteases of this family. All of the proteases described in this family have a similar amino acid sequence that can be expressed as two fragmented protein parts, coupled via short-acting interconnected peptides with inducible dimerization domains. Separate protease fragments form functional inactive proteins according to the invention. After addition of the ligand or another signal (e.g., light) which causes the association of the dimerization domains, structural and functional reconstitution of the split proteases is achieved, which cleave the next member of the signaling cascade containing the target peptide sequence for cleavage with the selected protease. Such a split protein may be a direct reporter (for example, luciferase or a fluorescence protein) or the next mediator of the signal cascade (for example, a transcription factor, kinase, phosphatase, or another protease), and its main feature is that its activity depends on cleavage.
A signaling system based on split proteases contains at least two split proteases. Each of the split proteases in the system may have its target protein (mediator or reporter), but two or more proteases may function on the same target protein if it contains various identifiable cleavage sites. The concept of the invention is presented graphically in Figure 1.
Transmission of the signal through split proteases may trigger or stop the transcription of selected genes or secretion of selected molecules, which is useful for the treatment of nervous system disorders, hormonal disorders, for metabolic and blood flow restrictions and other diseases. The invention is useful for controlling the action of human, animal or plant cells, for transmitting the signal to other cells, for secreting peptides, proteins and other molecules, to detect a combination of two or more chemical, light or mechanical signals.
Figure legends
Figure 1: A schematic diagram of a signal system with orthogonal split proteases associated with dimerization domains that can be activated via an inductor that may be intracellular or external. In the presence of an inducer, the inactive split protease is assembled into the active protease and cleaves the target protein substrate, which leads to a change in the target protein, which may be a reporter, a different protease, an inactive enzyme, etc. Each orthogonal split protease can have its own dimerization domains, thus various inductors can activate various orthogonal proteases, and in this way lead to different cell responses via logic operations,. Proteases break down the target protein that leads to the continuation of the signal pathway (for example, transcriptional regulation) or represents an output signal (for example a
fluorescent protein). If the target protein contains recognizable sites for cleavage with various proteases, the signal transfer with proteases can act as a logic circuit.
Figure 2: Orthogonality of selected proteases. The activity of each protease is shown as a cyclic luciferase activity with a marked cleavage site for the protease. The target sequences for cleavage of each protease are shown.
Figure 3: Inducibility of selected proteases. The activity of each protease is shown as cyclic luciferase activity with the appropriate cleavage site for the selected protease. A) Induction of split proteases with rapamycin. B) Induction of the split TEV protease with blue light. C) Induction of split PPV protease with blue light.
Figure 4: Activation of orthogonal signaling pathways mediated by TEV and PPV proteases. TEV protease cleaves the transcription factor, which increases the expression of the mCitrin reporter. PPV protease activates the cyclic luciferase reporter via cleavage.
Figure 5: The NOR logical operation mediated by TEV and PPV proteases. A) Schematic NOR logic circuit. Target proteins form an active luciferase reporter (output signal 1) in the absence of a signal (input signal 0 0) with dimerization through the wrapped helix P3 and AP4. In the presence of either or both of the TEV and PPV proteases (input signals 1 0, 0 1 and 1 1), the fragments of the reporter break off from the coiled coils and, therefore, no longer form a working reporter (output signal 0). B) The measured activity of the reporter of the NOR logical function with coexpression of TEV and PPV proteases. The addition of either protease or both proteases simultaneously reduces the activity of the reporter.
Figure 6: A of NIMPLY B logic function mediated by TEV split protease with light induction and PPV split protease by chemical induction. A) Schematic of the A NIMPLY B logic circuit. The P3mS peptide prevents the formation of a coiled coil between AP4 and P3 (input signal 0 0).When cleaved with TEVp protease, P3mS is removed and reconstitution of luciferase occurs (input signal 1 0). PPV protease cleaves the C -terminal luciferase fragment from the peptide P3, thereby preventing the enzyme reconstitution (input signals 0 1 and 1 1). B) The measured activity of the reporter of the A NIMPLY B logic function with different input signals. The activity of the reporter is high only when stimulated with light that
activates the TEV protease, but not with stimulation with rapamycin that activates PPV protease or with rapamycin and light at the same time.
A detailed description of the invention and applied examples
The invention relates to a combination of two or more orthogonal split proteases that recognize a target sequence comprising of at least 6 amino acids with dimerization domains that enable the functional reconstitution of protease activity, as well as target proteins with a target sequence recognized by the proteases.
The combination of orthogonal split proteases with dimerization domains includes, according to the invention, :
• At least two split proteases, each of which is expressed as a minimal of two split protein fragments and which are selected preferentially from the NIa family of potivirus proteases, preferentially proteins SuMMV, SbMV, PPV and TEV; preferentially at least one of the split proteases is composed of two proteins with the protein sequence of SEQ ID 4 and SEQ ID 6, SEQ ID. 10 and SEQ ID. 12, SEQ ID. 16 and SEQ ID. 18, SEQ ID. 22 and SEQ ID. 24, or their homologs that have at least 30% similarity of the amino acid sequence
• split proteases fused with dimerization domains that allow their assembly and are: (i) spontaneously assembled domains and / or (ii) their assembly is induced by intracellular or external physical, chemical or biological stimuli
• dimerization domains that assemble spontaneously and are selected from domains that spontaneously form dimers, preferentially dimeric proteins, helical coils and beta structures, all of which have, preferentially, orthogonal properties
• The dimerization of the domains is triggered by (i) an intracellular signal such as: a change in the concentration of a metabolite or a secondary signaling molecule, a change in the enzyme activity, or with (ii) an external signal such as temperature change, pH value, mechanical stress, change in osmotic pressure, ultrasound, light, chemical or biological signal
• dimerization domains, preferentially coiled helices, light inducible domains; preferentially CRY2 and CIBN, chemically inducible domains; preferentially FKBP and FRB, or calcium-dependent domains; preferably calmodulin and Ml 3, in fusion with split protease fragments that can form homodimers, heterodimers or which form a covalent bond as a result of self cleavage (inteins) and whose dimerization is dependent on the input signal, the input signal being a mechanical stimulus; preferably ultrasound or
touch, a light signal, a chemical ligand; preferably rapamycin, or another physiologically relevant signal, such as proteolytic cleavage
• dimerization domains that spontaneously assemble and are selected from orthogonal coiled coil pairs
• a native, recombinant or artificially generated target protein with a recognizable sequence for specific proteases, the recognizable sequence being at least 6 amino acid residues long
• at least one protein comprising at least one recognizable cleavage site for at least one of the split proteases and may form the next sequential step in a signaling pathway, for instance a transcriptional regulator, or which functions as a therapeutic target, a therapeutic protein or peptide or a reporter protein, preferably luciferase, SEAP or a fluorescent protein
• sufficiently long unstructured linker peptides between the individual protein domains that allow for the correct functional reconstitution of protease fragments,
• optionally includes any number of copies of localization signals, preferably a nuclear localization signal, a signal sequence for transport to the ER or transmembrane domains, and
• Optionally includes an arbitrary marker sequence for protein detection or affinity chromatography (e.g., HIS and AU1 markers).
The invention relates to cells that contain the above-described orthogonal signaling pathways and in which the signal path leads to a response that can be expressed in several ways, such as (i) release of endogenous cell metabolites, peptides or proteins (e.g., hormones), ( ii) releasing exogenous metabolites, peptides or proteins, preferably therapeutic proteins, (iii) modifying the activity of endogenous or exogenous proteins (preferably enzymes) in the cell, or (iv) controlling the expression of genes.
The invention relates to cells selected from bacterial or eukaryotic cells, plant cells or human cell lines, preferentially the invention relates to mammalian cells and human cell lines, for example to neurons or other nervous system cells, T lymphocytes or other immune cells or pancreatic beta cell.
Definitions
The term "split protease" refers to two or more polypeptides ("fragments") derived from the protease sequence, each of them being equal to one part of the whole protease. The individual fragments alone are not catalyticaly active. The formation of a proteolytically active complex requires the interaction of protease fragments; this process is termed "reconstitution". Protease fragments are selected so that reconstitution cannot occur between them without the aid of additional dimerization domains, which are expressed in fusion with protease fragments in the form of a chimeric protein. The term »chimeric
protein« means a protein or polypeptide consisting of sequences derived from non-native proteins and formed upon the translation of a chimeric nucleic acid that combines the records for the individual domains of non-native proteins composed to form a single open reading frame.
The term "dimerization domain" refers to protein domains that connect independently or in the presence of a ligand to one another through covalent or non-covalent interactions. The term "homodimerization" refers to domains that connect to other domains of the same type, and the term "heterodimerization" refers to domains that connect to other domains of a different type. The term "constitutive dimerization domains" refers to dimerization domains that connect themselves independently, such as, for example, coiled coils, and the term "inducible dimerization domain" refers to dimerization domains that merge only in the presence of a dimerization signal.
The term "signal" refers to a measurable change within a cell or in its environment. The term "input signal" refers to a change that triggers a signalling pathway, and the term "output signal" refers to a change that occurs as a result of the signalling pathway. An input signal may be part of an endogenous signalling pathway or is triggered exogenously and causes a physiological response of the cell, for example, a change in the concentration of a secondary signalling molecule, such as calcium, or a dimerization signal that affects the dimerization of endogenous or exogenous dimerization domains.
The term "dimerization signal" refers to ligands, light and mechanical stimuli, pH changes, post- translational modification of dimerization domains, change in the secondary signalling molecule concentration, or alteration of the transmembrane potential, which cause the dimerization of domains that do not have intrinsic affinity with each other and cannot be connected independently in the absence of a dimerization ligand or signal.
The term "mechanical signal" used herein refers to all kinds of mechanical disturbances / forces such as ultrasound, touch, osmotic stress and friction due to the flow of fluid that affect the cells or the cell membrane. The mechanical signal can be generated by the pressure of a particular solid object, liquid, or other cells on the cell membranes, due to the gravitational, centrifugal, shear or other direct force, or due to indirect forces that induce action on the cell membrane, such as osmosis, stretching or shrinking of the membrane due to temperature or other factors.
The term "ultrasound" refers to acoustic waveforms with a frequency between about 20 kHz to about 15 MHz, generated by a device with which the strength or sound amplitude, frequency in combination with a suitable converter, and a time dependency regime for ultrasonic pulses can be controlled.
The term »chemical signal« used herein refers to a change in ligand concentration. The term »ligand« used herein refers to short biopolymers, organic or inorganic molecules and ions that can bind to proteins, preferably to dimerization domains.
The term "light signal" refers to electromagnetic waves in the visible range (wavelengths between 400 and 700 nm), near infrared (wavelengths between 700 nm and 4 μπι) or ultraviolet (wavelengths between 10 nm and 400 nm), which may cause conformational or chemical changes in biological molecules, preferably dimerization domains or their ligands.
The term "signalling pathway" used herein refers to one or more events in a cell that may comprise ligand binding, dimerization of proteins or peptides, influx of secondary messenger molecules, alteration of the transmembrane potential, enzyme-catalyzed reaction or other cell changes, and which are triggered by an input signal, and which produces an output signal as its final product.
The term »orthogonal« used herein refers to the characteristic of the signalling pathways to take place separately, i.e., that the individual parts of one signalling pathway do not interact with parts of other endogenous or exogenous signalling pathsways. The main feature of the orthogonal signaling pathways is that the input signal of each path leads to the output signal independently of the presence or absence of other signalling pathways or their input and output signals.
The term »orthogonal proteases« means two or more proteases that differ in the order of the target substrate so that none of the orthogonal proteases do not split the target substrate of the second orthogonal protease.
The term "inducible protease" refers to the property of the protease, by which its enzymatic activity significantly changes in the presence of an input signal. The input signal can also be the output signal of a separate signalling pathway.
The term "linker peptide" refers to amino acid sequences whose role is to separate the individual domains of the assembled protein and to enable their proper spatial orientation. The role of the linker peptide in the fusion protein, the inclusion of which is optional, can also be used as a space to include the insertion of the cleavage site, the posttranslational modification site, the marker sequence, or the localization signal.
The term »cleavage site«refers to the amino acid sequence to which a specific protease can bind and within which the protein is cleaved by hydrolysis of the peptide bond. The term »marker sequences« refers to the amino acid sequences added to the protein for easier purification, isolation, or detection of the protein. The term localization signal« refers to an amino acid sequence that directs the protein to a
particular location in the cell. Localization signals vary according to the host organism in which the protein is expressed. The amino acid sequences of localization signals are well known among experts in the field and it is also known which signal sequence operates in specific organisms.
The position of linker peptides, marker sequences, cleavage sites, and localization signals is arbitrary, although they must be posited in a way that allows for the functional expression of the protein and preserves the functions for which these amino acid sequences have been selected and which are well known to experts in the field.
The term »cell« used herein refers to an eukaryotic cell, a cellular or multicellular organism (cell line) cultured as a single cell entity that has been used as a recipient of nucleic acids and includes the daughter cells of the original cell that has been genetically modified by the inclusion of nucleic acids. The term refers primarily to cells of higher developed eukaryotic organisms, preferably vertebrates, preferably mammals.
The term »cells« also refers to human cell lines and plant cells. Naturally, the descendants of one cell are not necessarily completely identical to the parents in morphological form and its DNA complement, due to the consequences of natural, random or planned mutations. "Genetically modified host cell" (also "recombinant host cell") is a host cell into which the nucleic acid has been introduced. The eukaryotic genetically modified host cell is formed in such a way that a suitable nucleic acid or recombinant nucleic acid is introduced into the appropriate eukaryotic host cell. The invention hereafter includes host cells and organisms that contain a nucleic acid according to the invention (transient or stable) bearing the operon record according to the invention. Suitable host cells are known in the field and include eukaryotic cells. It is known that proteins can be expressed in cells of the following organisms: human, rodent, cattle, pork, poultry, rabbits and the like. Host cells may include cultured cell lines of primary or immortalized cell lines.
The term »nucleic acids« used herein refers to a polymeric form of nucleotides (ribonucleotides or deoxyribonucleotides) of any length and is not limited to single, double or higher chains of DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids, or polymers with a phosphorothioate polymer backbone made from purine and pyrimidine bases or other natural, chemical or biochemically modified, synthetic or derived nucleotide bases.
The term »polypeptide«, »protein« , »peptide«, used herein refers to the polymeric forms of amino acids of any length. The term »functional polypeptide* used herein refers to a polypeptide form of any length of amino acids which expresses any function, for instance: structure formation, localizing to a
specific location, localizing to specific organelles, facilitating and triggering chemical reactions, binding to other functional polypeptides.
The term »heterologous« used herein refers to the context of genetically modified host cells and refers to a polypeptide for which at least one of the following claims applies: (a) the polypeptide is foreign ("exogenous") to the host cell (it is not naturally present within the cell); (b) the polypeptide is naturally present ("endogenous") in a given host microorganism or host cell, but is produced in an unnatural amount in the cell (more than expected or in greater amounts than found in nature) or is differentiated in the nucleotide sequence from the endogenous nucleotide sequence, so that the same protein (having the same or substantially similar amino acid sequence) as the endogenous protein is produced in unnatural amounts (more than expected or more than found in nature) in the cell.
The term »homologous« used herein refers to proteins or nucleic acids with a well-conserved amino acid or nucleotide sequences, preferably with at least 50% conservation and no less than 20% conservation, determined by protein or nucleic acid comparative techniques known to experts in the field. Homologous nucleic acids encode homologous proteins.
The term "recombinant" used herein means that a particular nucleic acid (DNA or RNA) is a product of various combinations of cloning, restriction and / or ligation leading to a construct having structurally coding or non-coding sequences different from endogenous nucleic acids in a natural host system. Generally, a DNA sequence encoding a structurally coding sequence may be formed from cDNA fragments, from short oligonucleotide linkers or from synthetic oligonucleotides from which a synthetic nucleic acid is obtained which can be expressed on a recombinant transcription unit in a cellular or cell- free transcription and translation system. Such a sequence can be used in the form of an open reading frame in which there is no interference from the internal non-translated sequences (introns) which are usually present in eukaryotic genes. Genomic DNA which contains important sequences can also be used to form a recombinant gene or transcription unit. Sequences of untranslated DNA may be present at the 5'- or 3'-end of the open reading frame, where such sequences do not affect the manipulation or expression of the coding regions and can act as modulators for the production of desired products through various mechanisms.
The insertion of the vectors into the host cells is carried out by conventional methods known from the field of science, and the methods relate to transformation or transfection and include: chemically induced insertion, electroporation, micro-injection, DNA lipofection, cellular sonication, gene bombardment, viral DNA input, as well as other methods. The entry of DNA may be of transient or stable.
Transient refers to the insertion of a DNA with a vector that does not incorporate the DNA of the invention into the cell genome. A stable insertion is achieved by incorporating DNA of the invention into the host genome. The insertion of the DNA of the invention, in particular for the preparation of a host organism having stably incorporated DNA of the invention, can be screened by the presence of markers. The DNA sequence for markers refers to resistance to antibiotics or chemicals and may be included on a DNA vector of the invention or on a separate vector.
The implementation examples that will be described in more detail are designed to best describe the invention. These descriptions have no intention of limiting the scope of the invention and its applicability, but are merely intended to provide a better understanding of the invention and its use.
Implementation examples
Example 1 : Preparation of cells, which feature a signaling pathway mediated by split proteases
Preparation of DNA records for orthogonal split proteases and their targets. In order to prepare DNA constructs, the inventors used molecular biology methods such as: chemical transformation of competent E. coli cells, plasmid DNA isolation, polymerase chain reaction (PCR), reverse transcription - PCR, PCR linking, nucleic acid concentration determination, DNA agarose gel electrophoresis, isolation of fragments of DNA from agarose gels, chemical synthesis of DNA, DNA restriction with restriction enzymes, cutting of plasmid vectors, ligation of DNA fragments, purification of plasmid DNA in large quantities. The exact course of experimental techniques and methods are well known to experts in the field and are described in the manuals of molecular biology.
All the work was performed using sterile techniques, which are also well known to the experts in the field. All plasmids, completed constructs and partial constructs were transformed into the bacteria Escherichia coli by chemical transformation. Plasmids for transfection into cell lines (animal, plant or human) have been isolated using a DNA isolation kit that removes endotoxins.
In the described cases, the proteases were selected from the proteases of the NIa family of potivirus proteins, specifically the SuMMV, SbMV, PPV and TEV proteases. Cleavage sequences for each of the described proteases are known to experts in the field. Based on the amino acid sequence similarity between these proteases and the already known sequence of split TEV protease, we prepared nucleotide transcripts for split proteases in fusion with dimerization domains. In the described cases, the light-induced domains of CRY2 and CIBN (Kennedy et al., 2010) and the rapamycin inducible domains of
FKBP and FRB were selected as dimerization domains (Sabatini et al., 1994; Brown et al., 1994; Sabers et al., 1995).
The sequences of split protease fragments according to the invention are listed in Table 1. These fragments alone do not form catalytically active enzymes. The final gene constructs of split proteases in fusion with dimerization domains that allow reconstitution into a functioning enzyme are listed in Table 2. The remaining sequences used to illustrate the invention are listed in Table 3. All operons were prepared by techniques according to methods known to experts in the field. The operones were inserted into plasmids suitable for eukaryotic systems. The suitability of the nucleotide sequence was confirmed by the inventors by sequencing and restriction analyzes.
Table 1: List of sequences of split proteases that were used to illustrate the invention and their recognizable cleavage sites
Table 2: Split proteases with dimerization domains that were used to illustrate the invention.
Seq Name Plasmid Construct description
ID. vector
25, FKBP-cTEVp pcDNA3 C- terminal part of the TEV protease in fusion with
26 the dimerization domain FKBP
27, FRB-nTEVp pcDNA3 N- terminal part of the TEV protease in fusion with
28 the dimerization domain FRB
29, FKBP-cTEVp- pcDNA3 C- terminal part of the TEV protease in fusion with
30 NLS the dimerization domain FKBP and a nuclear
localization signal
31, NLS -FRB-nTEVp pcDNA3 N- terminal part of the TEV protease in fusion with
32 the dimerization domain FRB and a nuclear
localization signal
33, FKBP-cPPVp pcDNA3 C- terminal part of the PPV protease in fusion with
34 the dimerization domain FKBP
35, FRB-nPPVp pcDNA3 N- terminal part of the PPV protease in fusion with
36 the dimerization domain FRB
37, FKBP-cSuMMVp pcDNA3 C- terminal part of the SuMMV protease in fusion
38 with the dimerization domain FKBP
39, FRB -nSuMMVp pcDNA3 N- terminal part of the SuMMV protease in fusion
40 with the dimerization domain FRB
41, FKBP-cSbMVp pcDNA3 C- terminal part of the SbMV protease in fusion with
42 the dimerization domain FKBP
43, FRB nSbMVp pcDNA3 N- terminal part of the SbMV protease in fusion with
44 the dimerization domain FRB
45, CRY2-nTEVp pcDNA3 N- terminal part of the TEV protease in fusion with
46 the dimerization domain CRY2
47, CIBN-cTEVp pcDNA3 C- terminal part of the TEV protease in fusion with
48 the dimerization domain CIBN
49, CRY2-nPPVp pcDNA3 N- terminal part of the PPV protease in fusion with
50 the dimerization domain CRY2
51, CIBN-cPPVp pcDNA3 C- terminal part of the PPV protease in fusion with
52 the dimerization domain CIBN
Table 3: Fusion proteins and operones which were used, appart from the split proteases, to illustrate the invention.
53, cycLuc_TEVs pcDNA3 Reporter protein cyclic luciferase containing a
54 cleavage site for the TEV protease.
55, cycLuc_PPVs pcDNA3 Reporter protein cyclic luciferase containing a
56 cleavage site for the PPV protease.
57, cycLuc_SbMVs pcDNA3 Reporter protein cyclic luciferase containing a
58 cleavage site for the SbMV protease.
59, cycLuc_SuMMVs pcDNA3 Reporter protein cyclic luciferase containing a
60 cleavage site for the SuMMV protease.
61, riLuc-TEVs-AP4 pcDNA3 N-terminal part of luciferase fused with the peptide
62 AP4 and labeled with a cleavage site for the TEV
protease
63, riLuc-AP4-TEVs- pcDNA3 N-terminal part of luciferase fused with the peptide
66 P3mS AP4 and P3mS and labeled with a cleavage site for
the TEV protease
67, P3-PPVs-cLuc pcDNA3 C- terminal part of luciferase fused with the peptide
68 P3 and labeled with a cleavage site for the PPV
protease
69, 6xN_Pmin_mCit pGL4.16 Reporter plasmid with 12 continuous TAL (N)
70 transcriptional factor binding sites in front of a
minimal promoter and the mCitrin reporter protein genome
70, TAL(N)-VP16- pcDNA3 TAL(N) transcription factor, that features a TEV
71 TEVs-KRAB protease cleavage site in between a VP 16 activation
domain and a KRAB repressor domain
Methods and techniques of cultivating cell cultures are well known to experts in the field and are therefore briefly described in order to illustrate the implemented examples. Cells from the HEK293T cell line were grown at 37 ° C and 5% C02. For cultivation, a DMEM medium containing 10% FBS was used, containing all the necessary nutrients and growth factors. When the cell culture reached an appropriate density, the cells were grafted into a new breeding flask and / or diluted. For the use of cells in experiments, the number of cells was determined by a hemocytometer and seeded in density 2.5xl04 per hole into the microtiter plate with 96 holes 18-24 hours before transfection. The seeded plates were incubated at 37 ° C and 5% C02 until the cells were 50-70% confluent and ready for transfection by transfection reagent. The transfection was carried out according to the instructions of the transfection reagent manufacturer (e.g., JetPei, Lipofectamine 2000) and was adapted for the microtiter plate used.
Example 2. Catalytic activity of split proteases in mammalian cells at stimulation with various signals and orthogonality of cleavage with individual protease
Cyclic firefly luciferase Flue based reporter system (Kanno et al. 2007) was used to detect catalytic activity of examined proteases. Cyclic luciferase, used as a reporter protein, was prepared by reorganizing amino acid sequence of firefly luciferase. Amino acids 4-233 from N-terminus were placed downstream to the C-terminus, connecting the two domains by a short linker which contains cleavage site for one of the following proteases: TEV, PPV, SuMMV or SbMV. The newly prepared protein was further modified by fusing two fragments of an intein via short linker to the ends, namely C-fragment of intein DnaE from organism Nostoc punctiforme was fused to N-terminus and N-fragment of intein DnaE from organism N. punctiforme to the C-terminus followed by amino acid sequence of peptide PEST-CL1 for fast digestion of the protein, previously known to experts from this field of science. Prepared luciferase based reporter thus forms inactive cyclic proteins, which can be cleaved with a target protease, resulting in structural reorganization and gain of luciferase catalytic activity.
HEK293T cells, implanted in 96-well plates, were transfected one day prior to the experiment with plasmids, which encode for one of examined proteases or two proteins, that combined form an active split protease; plasmids that encode previously described cyclic luciferase as a reporter protein and plasmid that encodes for luciferase from organism Renilla reniformis Rluc (GenBank AF362545.1). Cells were stimulated by addition of rapamycin to end concentration of 1 μΜ one day prior to measurement or with light beam (455 nm wavelength) in specially prepared machine 30min prior to measurement.
To analyze the activity of reporter proteins, cells were lysed with appropriate buffer according to instructions provided by the manufacturers (Promega). We measured catalytic activity of Flue and Rluc respectively. Rluc is expressed independently of all other components in our system and therefore provides the information of fraction of transfected cells. Flue presents catalytic activity of cyclic luciferase after protease cleavage. Ratio Fluc/Rluc (RLU - relative luciferase units) therefore tells us the normalized value of stimulated cells with respect to transfected cells.
Results:
It is clear from Figure 2 that each of the proteases TEV, PPV, SuMMV and SbMV efficiently cleaves cyclic luciferase with corresponding cleavage site, whereas the activity of proteases on cleavage sites for other proteases is negligibly low. In these experiments we used the full amino acid sequences of proteases.
Further we cleaved proteases TEV, PPV, SbMV and SuMMV in two fragments and fused them to ligand-binding domains FKBP and FRB. From figure 3 A we can clearly see that at addition of rapamycin split proteases TEV, PPV, SbMV and SuMMV fused to dimerizing domains FKBP and FRB cleave
luciferase with corresponding cleavage site. We also prepared fusion of split proteases TEV and PPV with proteins RCY2 and CIBN, which are known to dimerize at stimulation with blue light. From figures 3B and 3C we can conclude that split proteases TEV and PPV fused to dimerization domains CRY2 and BICN cleave cyclic luciferase with corresponding cleavage site when stimulated with blue light.
Example 3. Control of orthogonal signal pathways with split proteases and different input signals
To detect the activity of two orthogonal signaling pathways, we prepared protease-cleavage- dependentTAL (N) -VP16-TEVs-KRAB transcription factor in addition to cyclic luciferase. The transcription factor was prepared in such a way that the sequence for the VP 16 activation domain was aded at the C-terminus of the TAL DNA binding domain, which is well known to the field experts, followed by a binding peptide with a recognizable sequence for cleavage with the TEV protease and the repressive domain KRAB. The VP16 and KRAB domains are also well-known to the field experts. During the cleavage, the transcription factor changes the mode of action from repression to activation of the transcription of genes. To determine the activity of the transcription factor, a gene for the yellow fluorescence protein mCitrine was inserted into the plasmid after a minimal promoter with binding sites for the DNA-binding TAL domain.
The HEK293T cells, implanted in 96-hole plates, were transfected one day before the experiment with plasmids bearing cyclic luciferase code with a recognizable cleavage site with PPV protease, a transcription factor TAL (N) -VP16-TEVs-KRAB, mCitrin under the appropriate promoter and a code for luciferase Rluc, as well as plasmid with a TEV or PPV protease code as marked.
To analyze the activity of the mCitrin reporting protein, the media was removed from the cells and then measured by a fluorescence reader of SynergyMX plates (manufactured by BioTek) with excitation at the wavelength of 512 nm and an emission at the wavelength of 532 nm. The fluorescence of untransfected cells was subtracted from the value obtained. For the analysis of the reaction cyclic luciferaseprotein activity, the cells were lysed with buffer according to the manufacturer's instructions (Promega) and then the activity of FLuc and Rluc was measured as in the above experiment.
Result:
From Figure 4 it can be seen that, in the presence of TEV protease, only the activity of the luciferase rapporteur increases; in the presence of PPV protease only the activity of the fluorescence reporter increases. In the presence of both orthogonal proteases, the activity of both reporters is increased. This confirms that signal paths are orthogonal.
Example 4: Logical functions with signal transmission via proteases
Logical functions NOR and A NIMPLY B have been developed as examples of the logical circuit with the transmission of the signal with proteases, the latter being the equivalent composite logic function A AND NOT B.
For the NOR function (Fig. 5A), a split luciferase rapporteur was used, which is comprised of an N-terminal luciferase fragment linked via a cleavage site for the TEV protease with an AP4 peptide and a C -terminal luciferase fragment, which is coupled via a cleavage site for the PPV protease with the peptide P3. Peptides P3 and AP4 form a coiled coil, which allows for the reconstitution of luciferase fragments into an active enzyme. The splitting of this rapporteur with the TEV protease tears off the N-terminal luciferase fragment from the peptide AP4, thereby preventing the formation of a functional reporting enzyme. Similar cleavage with the PPV protease clears the C-terminal luciferase fragment from P3 peptide, thereby preventing the formation of a functional reporting enzyme. Therefore, the output signal (activity of luciferase) can be detected only in the absence of input signals.
The HEK293T cells in the 96-hole plate were transfected one day before the experiment with plasmids bearing the Rluc luciferase, the nLuc-TEVs-AP4 and P3-PPVs-cLuc luciferase, and plasmids with TEV protease and PPV protease as marked. The activity of the reporting proteins Flue and Rluc was measured as described above. Result:From Fig. 5B it can be seen that we observed a decrease in the activity of the reporter when coexpressed with any of the selected proteases. This is in line with the expected function of the NOR function.For the function A NIMPLY B (Fig. 6A), a split protease reporter was also used, but in this case it consists of an N-terminal luciferase associated with the peptide AP4, which is further linked to the P4mS peptide by the cleavage site for the TEV protease. A C-terminal luciferase fragment bound to the P3 peptide via the cleavage site for the PPV protease was added to the system as well. The peptides AP4 and P3mS form a coiled coil, which prevents the formation of a coiled coil between AP4 and P3 and thus the reconstitution of the functional luciferase enzyme. When the TEV reporter is split, the formation of a coiled coil between AP4 and P3mS destabilizes as it cleaves the connecting peptide between them. Since the peptide P3 forms a more stable coiled coil with AP4, now there may be reconstitution of luciferase, but only in the absence of PPV protease, since it cleaves the C- terminal luciferase fragment from P3 peptide. The outcome signal (activity of luciferase) can therefore be detected only when cleavage with TEV protease and absence of cleavage with PPV protease.
The HEK293T cells in the 96-hole plates were transfected one day before the experiment with plasmids bearing the Rluc luciferase, the nLuc-AP4-TEVs-P3mS and P3-PPVs-cLuc reporting protein, plasmids with split TEV with inducible CRY2 and CIBN domains, and plasmids bearing splice-protected PPV proteases with inducible FKBP and FRB domains. The cells were stimulated by the addition of rapamycin to a final concentration of 1 μΜ or with light with a wavelength of 455 nm 15 minutes before the measurement in a specially prepared device. The activity of the reporting Flue and Rluc protein was measured as described above.
Result:
From Figure 6B it can be seen that an increased activity of the reporter was observed only when the cells were stimulated with light, causing activation of the TEV protease, while in the absence of rapamycin which otherwise induces PPV protease activation. This is consistent with the expected function of A NIMPLY B function, where the input signals A and B are light and rapamycin.
Claims
1. A combination of two or more orthogonal split proteases which recognizes target sequence that covers at least 6 amino acids with dimerization domains which enable functional reconstitution of protease activity and target proteins with target sequence that is recognized by proteases.
2. A combination of orthogonal split proteases with dimerization domains according to claim 1 wherein combination is made from at least two orthogonal proteases selected from the family of Potiviridae proteases NIa, preferably from viruses TEV, PPV, SuMMV and SbMV.
3. A combination of orthogonal split proteases with dimerization domains according to claims 1 or 2 wherein at least one of the split protease is composed from two proteins with protein sequence SEQ ID 4 in SEQ ID 6, SEQ ID. 10 in SEQ ID. 12, SEQ ID. 16 in SEQ ID. 18, SEQ ID. 22 in SEQ ID. 24 or their homologues which have at least 30% amino acid sequence similarity.
4. A combination of orthogonal split proteases with dimerization domains according to claims 1 or 3 wherein split proteases are fused to dimerization domains which enable reconstitution and are: (i) domains that assemble spontaneously and/or (ii) their assembly is induced with an intracellular or extracellular physical, chemical or biological signal.
5. A combination of orthogonal split proteases with dimerization domains according to claims 1 or 4, wherein dimerization domains, which are domains that assemble spontaneously, are selected from domains that spontaneously form dimmers, preferably protein dimmers, coiled coils, beta structures, which have preferably orthogonal properties.
6. A combination of orthogonal split proteases with dimerization domains according to claims 1 or 5 wherein the assembly of dimerazation domain is triggered by (i) intracellular signal that is a change in metabolite concentration, a change in secondary metabolite, a change in enzyme activity or with (ii) extracellular signal that is change in temperature, pH, a mechanical stress, a change in osmotic pressure, an ultrasound, a light, chemical or biological signal.
7. A combination of orthogonal split proteases with dimerization domains according to claims 5 or 6 wherein at least one split protease is fused to dimerization domains FKBP and FRB or dimerization domains CRY2 and CIBN or M13 and CaM.
8. A combination of orthogonal split proteases with dimerization domains according to claims 1 or 7 wherein one part of split protease TEV or PPV si fused to dimerization domain CRY2 and the
other part of split protease is fused to CIBN which enables signal pathway activation in the presence of light signal.
9. A combination of orthogonal split proteases with dimerization domains according to claim 5 wherein dimerization domains which are domains that assemble spontaneously are selected from orthogonal coiled coils.
10. A combination of orthogonal split proteases with dimerization domains according to any one of claims 1 to 9 wherein the target protein with recognition sequence for protease is native, recombinant or synthetically generated protein.
11. A combination of orthogonal split proteases with dimerization domains according to any one of claims 1 to 10 wherein the target protein that contains recognition sequence for cleavage with at least one of the orthogonal split proteases is a protein for transcription activation.
12. A combination of orthogonal split proteases with dimerization domains according to any one of claims 1 to 10 wherein the target protein that contains recognition sequence for cleavage with at least one of the orthogonal split proteases is enzyme which activity depends on cleavage with the split protease.
13. A combination of orthogonal split proteases with dimerization domains according to any one of claims 1 to 10 wherein the target protein that contains recognition sequence for cleavage with at least one of the orthogonal split protease is a protein, a peptide or a hormone whose secretion from the cell depends on cleavage with split protease.
14. Cells from human, animals, fungi, plants or bacteria that express combination of orthogonal split proteases with dimerization domains according to any one of claims 1 to 13.
15. Cells according to claim 14 wherein said cells are human or animal cells, preferably neurons or other cells of the nerve system, T-lymphocytes, immune system cells, pancreas beta cells and said cells express combination of orthogonal split proteases with dimerization domains according to any one of claims 1 to 13.
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EP2895859B1 (en) * | 2012-09-12 | 2017-05-17 | The University of Queensland | Protease-based biosensor molecule |
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