JP2005527210A - Translocation-dependent complementarity for drug screening - Google Patents

Abstract

The present invention relates to various uses of protein fragments of complementary proteins to discover compounds or drugs that interfere with protein translocation / redistribution and / or protein-protein interactions. The present invention takes advantage of the fact that many interacting proteins are separated and located in distinct locations prior to the activation of the signaling pathway in which they exert their action. The present invention also utilizes interaction domains that are induced to interact (FRBP, FKBP12). Particularly disclosed is GFP (EGFP, F64L mutated EYFP) complementarity.

Description

Field of Invention

  The present invention relates to various uses of protein fragments of complementary proteins to discover compounds or drugs that interfere with protein translocation and / or protein-protein interactions. The present invention takes advantage of the fact that many interacting proteins are separated and located in distinct locations prior to the activation of the signaling pathway in which they exert their action. The present invention utilizes certain types of interaction domains that can interact by applying specific stimuli. By this means, complementary signals can be enhanced with respect to their complementary components. This component is guided to the same intracellular location following activation of the signaling pathway in which they act.

Background of the Invention

  Protein interactions with each other and with other cellular components are intrinsic to almost all cellular processes, which is especially true in intracellular signaling systems. Information typically passes through and between signal transduction systems through a series of such interactions.

  Existing methods for identifying interacting species can be divided into two groups, i.e .: only the task of bringing the slightly purified components in vitro together Methods such as surface plasmon resonance (evanescent wave method), protein mass spectroscopy, fluorescence correlation spectroscopy and anisotropy measurement, all common (I.e., the property that the component of interest has been separated from the cellular context). The second group includes all methods designed to work in living cells. Among them, many have been developed to work in yeast cells (yeast double hybrids, reverse yeast double hybrids and variants thereof), but some are applicable for use in mammalian cell systems. ing. Cellular methods for the detection of protein interactions have been described in the literature [Mendelsohn, A. et al. R. Brent, R.M. (1999) (Science 284 (5422): 1948)] provides a detailed review. Many of the methods are derived from the conventional double hybrid method. In these methods, transcriptional activity is achieved by bringing bi-partite transcriptional regulators into close proximity through the interaction of attached “bait” and “play” components. Be started. On the other hand, other methods rely on reconstitution of biochemical functions in vivo. Rossi et al. (2000) (Trends in Cell Biology 10: 119-122) has developed a protein interaction assay based on mammalian cells. In this assay, read-out is not transcriptional, but is a reconstitution of the mutated beta-galactosidase enzyme. Enzyme activity can be monitored upon reconstitution of the tetrameric enzyme. In addition, methods for monitoring protein interactions are based on optical readouts, ie fluorescence resonance transfer (FRET), or coincidence analysis (variation of fluorescence correlation spectroscopy), or It is a fluorescence lifetime change (fluorescence lifetime changes). The last three categories are commonly applied under simplified in vitro conditions, but attempts have been made to move them to the more complex environment of living cells.

  It has been suggested that the reassembly of specific enzyme fragments of a complete enzyme be used as a protein-protein interaction. Johnson and Varshavsky (Johnson, N., Varshavsky, A. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 10340-10344) disclose the reconstitution of ubiquitin. This reconstitution is detected through irreversible cleavage by the fusion ubiquitin protease and release of the reporter. In contrast to the double hybrid technology, this technology has the potential to monitor protein-protein interactions as a function of time, at the natural sites of the interaction in living cells.

  A similar system has been suggested for the reconstitution of other proteins, including β-galactosidase (Rossi, F., Charleston, CA, Blau, HM (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 8405-8410), dihydrofolate reductase (DHFR, W098 / 34120), and lactamase (Wehrman, T., Kleveland, B., Her, J. H., Balint, R. F.). Blau, HM (2002) Proc. Natl. Acad. Sci. U.S.A. 99, 3469-3474). The basic concept is that the function is lost by splitting a functional protein into two fragments. The two fragments are fused in-frame to proteins X and Y, respectively, and transformed or transfected into cells. Binding between proteins X and Y brings the two fragments together, thereby increasing the local concentration of complementary fragments, inducing folding of these fragments, and activity (similar to the activity of unfragmented proteins) Produces a functional protein having If the function is DHFR activity, the cells only survive when proteins X and Y bind to each other.

  Recently it has been described to use a somewhat similar system for assisted reconstitution and folding of fragments of fluorescent proteins. Since the function is fluorescence, the cells emit light upon excitation only when protein X and protein Y bind to each other, thereby assisting in complementation. Ghosh (I. Ghosh, A. D. Hamilton, L. Regan (2000) J. Am. Chem. Soc. 122, 5658-5659, W001 / 87919) is a GFP variant called sg100 (F64L, S65C, Q80R, Y151L, I167T and K238N). The present GFP has a single fluorescence excitation and emission peaks at 475 nm and 505 nm, respectively (similar to sg25 described by Palm (Palm, G. J., Zdanov, A., Gaitanaris, GA, Stauber, R., Pavakis, GN, Wlodwer, A. (1997) Nat. Struct. Biol. 4, 361-365)]. The complementation of a functional GFP fragment is two independent peptides, the first 157 N-terminal amino acid of this GFP (NtermGFP157) and the remaining 81 C-terminal amino acid of this GFP (starting from residue 158) Each of the GFP peptide fragments is fused with an interacting leucine zipper peptide that interacts with the fragment.

  Protein recruitment or re-localization plays an important role in many important signal transduction systems. This matter is evident in the following (i) to (iii): (i) Activation of protein kinase C (PKC), in this activation, supplementation to the plasma membrane is the natural process of activation of the enzyme. (Ii) activation of p42 / 44 MAP kinase depending on multiple proteins (translocation of these proteins from cytosol to membrane site has a central role); and (iii) The cyclic AMP-driven relocalization of rap1 is critical for its activation. The use of protein translocation in connection with the identification of potential medicines is disclosed in W098 / 45704. However, there is still a need for alternative techniques for detecting protein localization and any relocalization in intact intact cells. Current detection techniques are typically characterized by their low throughput and the need for expensive detection equipment.

Summary of the Invention

  The present application discloses that when expressed as two independent protein halves, certain GFPs are reconstituted to form a functional fluorescent protein. For example, when EGFP is expressed in mammalian cells, strong fluorescence is produced by selecting a split site located in the loop region between residues that form the beta sheet structure of the GFP β-barrel ( Example 5 and Example 7). Furthermore, this application describes that EYFP is also reconstituted, and surprisingly the fluorescence from the reconstituted protein is significantly enhanced (if it has the F64L mutation) (Examples). 9).

  The model GFP complementation system (using components that can be created to conditionally interact) responds to interactive stimuli in a dose-dependent manner as expected and, importantly, is almost spontaneous Can be used to detect compounds that block the conditional interaction of proteins fused with the complementary halves of the protein such that no rearrangement (complementarity) is observed (eg, GFP complementation system) (Example 11) This application demonstrates that.

  For the proteins that interact spontaneously, the examples disclosed herein disclose that the complementation system can be designed to report the degree of interaction between those proteins in living cells. Yes. This allows the two proteins to be kept in separate locations on the cell, thereby allowing the interaction to occur only after the translocation of one or both proteins, allowing them to be separated into one compartment within the cell. (Compartment) or proximity in location.

  One aspect of the present invention is constructed on the basis of the following concept; that is, signaling proteins, which interact with each other when active, are usually separate and spatially separated. Spontaneous reconstitution of complementary systems that exist in intracellular locations prior to activation of the signaling pathway they act on, and therefore are used to monitor their protein interactions, is avoided. This is the concept. This is clearly described in Example 14; here, induction of translocation from one local site to another causes the N and C termini of the complementary protein to reconstitute and function. (Fluorescence in this case). A similar system is described in Example 16; here constitutively interacting proteins are replaced with proteins of induced interaction.

Detailed disclosure

The present invention is achieved based on knowledge of the localization of proteins in the cell, which is used to bring the two halves of the enzyme in a controlled manner until they are in close proximity to the original back to the back. It is something to keep apart. Accordingly, a major aspect of the present invention relates to a cell, the cell comprising at least the following:
A first conjugate comprising an N-terminal fragment of the first protein and the complementary protein; and
A second conjugate comprising a C-terminal fragment of a second protein and a complementary protein;
Here, the first conjugate has a preferential position at a cell position that is separate and spatially separated from a position where the second conjugate is preferentially located,
Where the conditions are allowed, the first protein and the second protein bind to each other; and
Here, the complementary protein changes when the two fragments of the complementary protein move to a close position and the two fragments of the complementary protein form a fully functional protein ( altered characteristics).

  One use of the present invention allows one to identify a drug, which can block the interaction between the first protein and the second protein. One important property of the present invention to avoid such pre-drug interaction is to keep the two conjugates at different cell locations. Most (but not all) proteins are in equilibrium between various states. In such a relationship, this equilibrium provides a balance between the presence at one location and the presence at another location within the cell. For the purposes of the present application, two different cell locations are understood as preferential or preferred locations of the two proteins, so that no interaction between the two proteins occurs, i.e. they This is a condition for the fact that the meeting is not facilitated. If the balance between the two positions is greatly shifted to one, the protein will be preferentially present in one of those positions. Activation of the system as used herein initiates translocation, results of any treatment of the system; or forms complementarity of the two conjugates, ie, activation of the system is at least one of the proteins It means the result of any other cell signaling process that shifts one equilibrium from a different cellular location than the other protein to the same cellular location as the other protein. As an example, PKCβ is preferentially found in the cytoplasm, but not exclusively, whereas the interaction partner protein (RACK1) is preferentially localized in the plasma membrane (plasma membrane). Yes. Upon application of a stimulus (eg, phorbol ester PMA), PKCβ proteins are preferentially localized to the plasma membrane, where they are in close proximity to RACK1. Another example of a signaling protein that translocates to and from the plasma membrane is the STAT transcriptional regulator and AKT / PBK kinase. Examples of signaling proteins that translocate between the cytoplasm and nucleus are the NF kappa B transcription factor, androgen receptor, MAP kinase p38, ERK and JNK, MAPKAP kinase 2, and Forkhead transcription factor.

  Thus, for most uses of the present invention, the cellular location of the first conjugate is determined by the nature of the first protein. Similarly, the cell location of the second conjugate is determined by the nature of the second protein.

Currently known complementary proteins produce irreversible complementarity, because the correctly folded complex of complementary fragments is energetically favorable, i.e., if the folded fragment dissociates (if at all) This is because they dissociate very slowly. Thus, if the protein is in the same position and complementarity begins (this is due to spontaneous binding between the first protein in the first conjugate and the second protein in the second conjugate). Yes, small fractions of proteins at unfavorable positions will bind irreversibly. When bound irreversibly, it is no longer involved in the equilibrium of free protein conjugates between unfavored and favored positions. Ultimately, most or all of the protein conjugate is drawn by equilibration to the unfavorable position. This is called a sink effect. One way to minimize the spontaneous association between two proteins is for one of the proteins to interact with an anchor protein (which strictly limits its mobility within the cell). . In this case, one protein is fixed at one localization site, and the association between complementary fragments only occurs when the other protein moves to the anchored position.
In this aspect of the invention, the second conjugate further comprises an anchor protein, and the anchor protein anchors to a cell compartment different from the position where the first protein is preferentially located.

Another use of location information (where a constitutively interacting protein is released until the two conjugates are deliberately brought together) is to measure the translocation of the protein. This can be done in a particular cell, which comprises at least the following:
A first conjugate comprising a protein, interaction partner A and a first terminal fragment of a complementary protein; and
A second conjugate comprising an interaction partner B and a second terminal fragment of the complementary protein;
Here, the first conjugate has a preferential position at a cell position that is separate and spatially separated from a position where the second conjugate is preferentially located,
Here, the complementary protein changes when the two fragments of the complementary protein move to a close position and the two fragments of the complementary protein form a fully functional protein ( alternative charactaristics), and where said interaction partner A and interaction partner B bind to each other.

  In such cells, translocation of the protein thereby the first conjugate to the same cellular location as the second conjugate results in complementation of the first and second terminal fragments of the complementary protein.

  One method for minimizing the effect of spontaneous complementarity between two proteins (sinking effect, which produces an unwanted background signal) is the interaction partners A and B [which are Binding only when a specific interaction stimulus is applied (conditional interaction partner). In this way, only when an interaction stimulus binds the interaction partner, spontaneous meeting between the proteins generates the complementarity of the two complementary fragments, thereby making the complementary fragment functional. In close proximity with respect to the reassociation and formation of various proteins. In this embodiment, interaction partner A and interaction partner B bind to each other only when an interaction stimulus is applied.

  Obtaining further control of the position of the conjugate and minimizing the spontaneous association between the two conjugates means that one of the conjugates interacts with the anchor protein (this is intracellular Strictly restricts its mobility in In this case, one protein is fixed at one localization site, and the association between complementary fragments only occurs when the other protein moves to the anchored position. In this aspect of the invention, the second conjugate further comprises an anchor protein, and the anchor protein anchors to a cell compartment different from the position where the first protein is preferentially located.

  In one aspect of the invention, the first terminal fragment of the complementary protein is the N-terminal fragment of the complementary protein and the second terminal fragment of the complementary protein is the C-terminal fragment of the complementary protein. In another embodiment of the invention, the first terminal fragment of the complementary protein is a C-terminal fragment of the complementary protein and the second terminal fragment of the complementary protein is an N-terminal fragment of the complementary protein.

  The simplicity of the present invention is derived in part from complementary proteins.

  When two fragments of complementary proteins move to a close position and the two fragments of complementary proteins form a fully functional protein, the complementary proteins exhibit altered characteristics The fact then facilitates the detection of close positional relationships between two fragments of complementary proteins.

  In principle, any detectable property of a combined mass (measurablely different from that of separate composition parts) can be used as a signal of complementarity for those separate parts. Such properties include biophysical properties such as rotational or translational diffusion coefficients, fluorescence properties such as peak excitation or emission wavelengths, or Fluorescence lifetimes are included, but are not limited to these. Also, potentially useful and detectable properties include fluorescence or luminescence of more biochemical features such as catalytic efficiency or enzymatic activity, or the polypeptide sequence unique to one of the complementary partners. ) Characteristics related to conversion efficiency to a form that has acquired the ability to issue. Examples of proteins that can be made to acquire catalytic activity when the compositional portion is configured through complementarity include ubiquitin (Johnsson, N., Varshavsky, A. (1994)), β-galactosidase (Rossi, F et al (1997)) and β-lactamase (Wehrman, et al (2002)).

  The following describes details on how GFP can be separated to form two complementary fragments, such data is not available on the priority date of the present invention.

  However, as will be apparent to those skilled in the art, other proteins can be separated as well.

  A non-fluorescent fragment of a fluorescent protein that can be combined to form a functional fluorescent unit can be obtained by splitting the encoded nucleotide sequence of one fluorescent protein at the appropriate site, and each nucleotide sequence fragment independently ( It is usually produced by expressing as complementary proteins N-terminal and C-terminal fragments (here GFP) of complementary proteins.

  In one embodiment of the present invention, the complementary protein is a fluorescent protein (eg, green fluorescent protein (GFP)).

  Green fluorescent protein (GFP) is a protein of 238 amino acids in length, and is derived from Aequorea Victoria (SEQ ID NO: 1). However, the fluorescent protein has been isolated from other members of the gastrointestinal animal, for example, Discosoma sp. Red fluorescent protein from (Matz, M.V. et al. 1999, Nature Biotechnology 17: 969-973), GFP from Renilla reniformis, GFP from Renilla Muelleri, or from other animals, fungi or plants It is a fluorescent protein. GFP is a blue fluorescent variant of GFP disclosed in various modified forms [Heim et al. (Heim, R. et al., 1994, Proc. Natl. Acad. Sci. 91: 26, pp 12501-12504]. This is a Y66H variant of wild-type GFP; a yellow fluorescent variant (YFP) of GFP with S65G, S72A, and T203Y mutations (W098 / 06737); cyan fluorescence of GFP Variant (CFP) with Y66W color mutation and optionally F64L, S65T, N146I, M153T, V163A folding / solubility mutation (Heim, R., Tsien, RY (1996) Curr. Biol. 6, 178-182). The most widely used variant of GFP is EGFP, which has F64L and S65T mutations (WO 97/11094 and W096 / 23810) and an insertion of one valine residue after the first Met. . The F64L mutation is an amino acid upstream of position 1 from the chromophore.

  GFP with this folding mutation provides an increase in fluorescence intensity when GFP is expressed in cells at temperatures above about 30 ° C. (WO 97/11094).

  It is known that fluorescence in wild-type GFP depends on the presence of a chromophore, which is the cyclization and oxidation of SYG at positions 65-67 (in the expected primary amino acid sequence). As well as SHG sequences in other GFP analogs (positions 65-67) for the same reason.

  This example illustrates whether the fluorescence intensity from a clearly reconstituted protein is enhanced in GFP with the F64L mutation (as compared to GFP without this mutation). Accordingly, it is preferred that the GFP has an F64L mutation, by selecting a GFP having this mutation (eg, EGFP) or introducing this mutation into a special GFP (eg, YFP described in Example 9). Made.

  In the GFP nomenclature, “E” is placed in front of GFP (EGFP, EYFP, ECFP) so that this particular GFP has a codon usage optimized for mammalian cells Indicates that it is encoded by a nucleic acid. Many of these proteins also have an extra valine residue inserted after the initiating methionine residue (Met). This extra valine residue is not considered for residue numbering. As described above, in a preferred embodiment, the GFP of the present invention is selected from the group consisting of EGFP, EYFP, ECFP, dsRed, and Renilla GFP.

  In some of the examples of this application, EGFP is used. Thus, in a preferred embodiment of the invention, the GFP is EGFP. However, Example 9 and Example 11 show that EYFP has certain advantages. Thus, in another preferred embodiment of the invention, the GFP is EYFP. It is also shown that EYFP mutated at position 1 preceding the chromophore (E [F64L] YFP) has certain advantages. Thus, in a preferred embodiment, the GFP is E [F64L] YFP, or the GFP is E [S72A] GFP (which is F64L, S65T, S72A GFP).

One aspect of the present invention relates to the cell described above, wherein the N-terminal fragment of the complementary protein comprises an N-terminal fragment of GFP (a continuous stretch of amino acids from amino acid number 1 to amino acid number X of GFP). Where the peptide bond between amino acid number X and amino acid number X + 1 is within the loop of GFP, and the C-terminal fragment of the complementary protein is the C-terminal fragment of GFP (from amino acid number X + 1 to amino acid number 238 of GFP). A continuous stretch of amino acids up to).
In the present invention, wild-type GFP (SEQ ID NO: 1) (Chalfie, M., Tu, Y., Euskirchen, G., Ward, WW, Prasher, DC (1994) Science 263, 802-805 , This variant of GFP has a histidine residue at position 231). Crystal structure of GFP (Yang, F., Moss, LG, Philips, GN (1996) Nat. Biotech. 14, 1246-1251), presented in FIG. 5, Table 1 and Examples Based on the data, it is clear that the split in most arbitrary loops reconstructs after proper spatial access to complementary fragments assisted by conjugated protein interactions. For the purposes of this application, the term “loop” is understood as an irregular secondary structure turn or element.

Thus, in one aspect, the complementary proteins are two GFP fragments,
Where X is 7, 8, 11 or 12, preferably X is 9 or 10 in the Thr9-Val11 loop; or
Where X is 21, 22, 25 or 26, preferably X is 23 or 24 in the Asn23-His25 loop; or
Where X is 36, 37, 40 or 41, preferably X is 38 or 39 in the Thr38 to Gly40 loop; or
Where X is 46, 47, 56 or 57, preferably X is between 48 and 55, ie X is 48, 49, 50, 51, 52, 53, 54 or 55 in the Cys48-Pro56 loop? Or
Where X is 70, 71, 76 or 77, preferably X is between 72 and 75, ie, X is 72, 73, 74 or 75 in the Ser72-Asp76 loop; or
Where X is 79, 80, 83 or 84, preferably X is 81 or 82 in a His81-Phe83 loop; or
Where X is 86, 87, 90 or 91, preferably X is 88 or 89 in the Met88-Glu90 loop; or
Where X is 99, 100, 103 or 104, preferably X is 101 or 102 in the Lys101 to Asp103 loop; or
Where X is 112, 113, 118 or 119, preferably X is between 114 and 117, ie X is 114, 115, 116 or 117 in the Phe 114 to Thr 118 loop; or
Where X is 126, 127, 145 or 146, preferably X is between 128 and 144, ie X is 128, 129, 130, 131, 132, 133, 134, 135, 136, 137 in the llle128 to Tyr145 loop. 138, 139, 140, 141, 142, 143, 144; or
Where X is 152, 153, 160 or 161, preferably X is between 154 and 159, ie X is 154, 155, 156, 157, 158 or 159 in the Ala 154 to Gly 160 loop; or
Where X is 169, 170, 175, 176, preferably X is between 171 and 174, ie X is 171, 172, 173 or 174 in the llle 171 to Ser175 loop; or
Where X is 186, 187, 197 or 198, preferably X is between 188 and 196, ie X is 188, 189, 190, 191, 192, 193, 194, 195 or 196 in the lle188 to Asp197 loop Or
Where X is 208, 209, 215 or 216, preferably X is between 210 and 214, ie X is 210, 211, 212, 213 or 214 in the Asp210-Art215 loop.

Table 1. GFP secondary structure, GFP wild type sequence amino acid numbering. α and β represent α-helix and β-sheet secondary structures, respectively.

  Based on the findings disclosed in the Examples, it can be concluded that the appropriate split site in GFP is located in the loop region between residues that form the β sheet structure of the GFP β barrel. Accordingly, the division in GFP is preferably asn23-His25 loop, Thr38-Gly40 loop, Lys101-Asp102 loop, Phe114-Thr118 loop, lle128-Tyr145 loop, Ala154-Gly160 loop, lle171-Ser175 loop, lle188-Asp197 loop. Or in the Asp210-Arg215 loop (Table 1, FIG. 5).

  The data in this example clearly shows that the Ala154-Gly160 loop is very well adapted for GFP reconstruction. This is especially the case when GFP is separated between amino acids Q157 and K158 (ie when X is 157). Accordingly, a preferred embodiment of the invention relates to two GFP fragments, where X is 157 in the Ala154-Gly160 loop.

  The data of this example also describes that the lle171-Ser175 loop is very useful for GFP reconstruction. This is especially the case when GFP is separated between amino acids E172 and D173 (ie when X is 172). Accordingly, a preferred embodiment of the present invention relates to two GFP fragments, where X is 172 within the lle171-Ser175 loop.

  One alternative aspect of the present invention relates to a cell as described above, wherein the N-terminal fragment of the complementary protein is an N-terminal fragment of GFP (of amino acids 1 to X of GFP). Wherein the peptide bond between amino acid number X and amino acid number X + 1 is within the loop of GFP, and the C-terminus of said complementary protein is the C-terminal fragment of GFP (amino acid of GFP). With a continuous stretch of amino acids from number Y + 1 to amino acid number 238, where Y <X results in an overlap of two GFP fragments, where the peptide bond between amino acid Y and amino acid Y + 1 is GFP In the loop.

  As described in Example 9, fragments with overlapping sequences have certain advantages. Accordingly, one aspect of the invention relates to two GFP fragments, the fragments comprising: (a) an N-terminal fragment of GFP (comprising a continuous stretch of amino acids from amino acid number 1 to amino acid number X of GFP) Where the peptide bond between amino acid number X and amino acid number X + 1 is within the loop of GFP), and (b) a C-terminal fragment of GFP (a sequence of amino acids from amino acid number Y + 1 to amino acid number 238 of GFP) With stretch, where Y <X results in the overlap of two GFP fragments, where the peptide bond between amino acid Y and amino acid Y + 1 is within the loop of GFP).

  These overlapping GFP fragments are, for example, functional cloning systems where the highly flexible linker sequence is due to the very diverse nature of the fusion partner structure. It is very attractive in cloning systems that are needed. The overlapping fragment allows to have a long linker sequence in any of the fusion partners.

  With respect to the purpose of determining the Y identity in the C-terminal fragment of GFP as defined above, the same considerations apply as in the discussion of the value of X.

  In one aspect of the invention, the overlap is only a few amino acid residues, eg, XY = 1, XY = 2, XY = 3, XY = 4, XY = 5, XY = 6, XY = 7, XY = 8, XY = 9 or XY = 10.

  Due to the folding properties of GFP folding, a preferred embodiment of the present invention relates to the overlap of N-terminal and C-terminal fragments of GFP, where a peptide bond between amino acid Y and amino acid Y + 1 and between amino acid X and amino acid X + 1. The peptide bond in is within the loop of GFP. The resulting overlap is a complete α-racene or β-sheet secondary structure. In a typical embodiment of the present invention, the conjugated protein [wherein the N-terminal fragment of the complementary protein (the first terminal fragment of the complementary protein) is conjugated with the protein of interest] is A linker sequence is further provided between the complementary protein fragment and the corresponding protein of interest.

  The length and composition of the linker must be selected depending on the protein that the practitioner wishes to conjugate with the complementary protein fragment. The main function of the linker is to provide flexibility and avoid complementary steric hindrance. Interacting proteins should be free to interact and simultaneously complementary fragments should be free to fold. This is facilitated by a long linker sequence (eg, a linker of 20 or more residues). However, short linker sequences keep complementary protein fragments in close proximity to each other, i.e., increase the local concentration of complementary fragments and reduce the entropy cost of complementation. Access to information about the structure of interacting or homologous proteins and to information about interactions between interacting proteins is valuable because it allows 5-10 residues (protein interactions or complementarity). This is because it can facilitate the design of short linkers that do not distort. It is advantageous to fuse complementary protein fragments to the ends of interacting proteins, which are attracted closest during the interaction. However, excellent complementarity is observed even when 15-20 residue linker sequences are used and the ends of the interacting proteins are separated by more than 40 Å.

  Said linker should mainly consist of specific amino acid residues (ie small, hydrophilic, non-reactive and constitute poor protease cleavage sites, eg Ser, Thr, Gly) It is.

  A preferred aspect of the present invention relates to nucleic acids encoding any conjugates, fragments or fusion proteins as described above. In one embodiment, the nucleic acid construct encoding any protein according to the invention described above is a DNA construct. In another embodiment, the nucleic acid construct encoding any protein according to the invention described above is an RNA construct.

  As will be apparent to those skilled in the art, the conjugate can be constructed by by choice, and the protein is conjugated at the N-terminus or C-terminus with the first end of the complementary protein. . However, as described in the Examples, conjugation of the subject first protein to the N-terminal fragment of GFP is preferably performed on the C-terminus of the N-terminal fragment of GFP. Similarly, conjugation of the second protein of interest with the C-terminal fragment of GFP is preferably performed on the N-terminus of the C-terminal fragment of GFP.

  As is clear from this example, the protein of interest is a protein, a peptide or a non-proteinaceous partner.

  Thus, one aspect of the invention relates to a particular cell, in which the N-terminal fragment of the complementary protein is fused in frame with the first protein of interest.

  One aspect of the invention relates to a particular cell, wherein the first protein is fused to the N-terminus of the N-terminal fragment of the complementary protein.

  One aspect of the invention relates to a particular cell, wherein the first protein is fused to the C-terminus of the N-terminal fragment of the complementary protein.

  One aspect of the invention relates to a particular cell, in which the C-terminal fragment of the complementary protein is fused in frame with the second protein.

  One aspect of the present invention relates to a particular cell, wherein the second protein of interest is fused to the N-terminus of the C-terminal fragment of the complementary protein.

  One aspect of the invention relates to a particular cell, wherein the second protein of interest is fused to the C-terminus of the C-terminal fragment of the complementary protein.

  One embodiment of the present invention relates to a specific cell, wherein the N-terminal fragment of the complementary protein (fused with the first protein in frame) further comprises a linker sequence and an N-terminal fragment of the complementary protein. And the first protein.

  One embodiment of the present invention relates to a specific cell, wherein the C-terminal fragment of the complementary protein (fused with the second protein in frame) further comprises a linker sequence and a C-terminal fragment of the complementary protein. And the second protein.

  One aspect of the present invention relates to a specific cell, wherein the GFP is EYFP (further having a F64L mutation), X is 172, and the first protein (fused with the N-terminal fragment of GFP). Is fused to the C-terminus of the N-terminal fragment of GFP and the second protein (fused with the C-terminal fragment of GFP) is fused to the N-terminus of the GFP C-terminal fragment.

  One embodiment of the present invention relates to a specific cell, wherein said GFP is EYFP (further having a F64L mutation), X is 157, and said first protein (fused with an N-terminal fragment of GFP). Is fused to the C-terminus of the N-terminal fragment of GFP and the second protein (fused with the C-terminal fragment of GFP) is fused to the N-terminus of the GFP C-terminal fragment.

  It is not essential that the conjugate is expressed in the cell, but it is preferred that the first conjugate is expressed in the cell. Similarly, in a preferred embodiment, the second conjugate is expressed in the cell.

  In a series of embodiments of the invention, at least one of the conjugates has an anchor protein. In the context of the present invention, anchor protein means any and all cellular components, preferably genetically codeable cellular components, having a defined cellular distribution.

  When choosing the anchor, it is important to consider the nature of the system. By way of example, some proteins need to be phosphorylated or dephosphorylated by enzymes that are sequestered in the plane of the plasma membrane; for such proteins of interest, they are confined to the plasma membrane Selecting an anchor component that would be expected to be, and appropriately modifying the interacting protein in measuring protein-protein interactions, or allowing measurement of translocation to that position, Is appropriate. Thus, in one embodiment, a suitable anchor component that targets the conjugate to the plasma membrane comprises the transmembrane domain of epidermal growth factor receptor (EGFR) or a membrane of a particular protein of the integrin protein family A protein that contains a penetrating domain or that contains a c-Src myristoylated sequence (residues 1-14).

  In another embodiment, a histone protein is used as the anchor, or a protein that is normally restricted to the nucleolus (eg, p120 nucleoprotein) is used to direct the anchoring conjugate to the nucleus, Makes it possible to measure protein-protein interactions in the nucleus or to measure translocation to the nucleus.

  In another embodiment, the anchor protein is selected from proteins that are normally confined to the outer or inner membrane of the mitochondria (eg, VDAC, Fo subunit of ATP-ase, or NADH dehydrogenase). In another embodiment, the anchor protein is selected from the group of proteins that are normally restricted to a variety of different regions of the Golgi apparatus (eg, TGN38 or ADAM12-L). In another embodiment, the anchor protein is selected from the group of proteins usually restricted to focal binding complexes (eg, P125, FAK, integrin alpha or beta, or paxillin). In another embodiment, the anchor protein is a group of proteins normally associated with cytoskeletal structures (eg, F-actin chains or microtubule bundles such as MAP4, actin binding domain of alpha actinine (actinPaint), kinesin, myosin or dynein ) Is selected.

  In another embodiment, the anchor protein is a protein normally associated with a nuclear material or a nuclear component [eg, histone proteins (including histones H1, H2A, H2B, H3, H4, and variants thereof)] It is.

  In another embodiment, the anchor protein is a nuclear membrane (eg, A and B type lamin), or a nucleolus (eg, splicing body, cajal bodies, PML nucleolus (PML oncogenic domain), PODS)) or a transcription engine (eg, RNA polymerase POL-II).

  As stated, some configurations of the present invention require spontaneous interaction proteins. Examples of protein domains that can be used to generate spontaneous interactions are generally leucine zippers and coiled coil domains. These domains have been identified in many different proteins, and self-association properties have been predicted with high accuracy from linear sequences of amino acids.

  Examples of proteins with leucine zippers are c-Jun and c-Fos. Coiled-coil domains and other self-interacting domains are found in Rho kinases ROCK, Bcr, Bcr-Abl, all known AKAPs and in the regulatory subunit PKA. Other spontaneous binding proteins are the binding of JNK to the delta region in c-Jun and the binding of beta-catenin to the Tcf-4 transcription factor.

  The special utility of stimulus-induced interactions is distinguishable in the following: in the same cell, only very weak interactions previously existed or where no interaction existed ( distintive) it is possible to switch on the interaction. This ensures in advance that the distinguishable interaction is purely the result of a specific interaction between the two interaction partners. In the case of a specific conjugate (the conjugate is a specific anchor protein, including an anchor protein that positions the immobilized conjugate at a specific cell location), the use of stimulus-inducible interactions is That is, it ensures that this interaction provides a signal that can be measured by an assay instrument configured to detect the expected position of the complementary product.

  In embodiments with interaction partners A and B, the specificity and utility of their aspects has measurable affinity for each other in the absence of interaction stimuli. Not dependent on interaction partner A and interaction partner B. Thus, in one aspect, interactors A and B are selected in the appropriate pair of proteins targeted by immunosuppressive factors (eg, but not limited to, cyclosporin A, rapamycin and FK506). . These proteins include, but are not limited to, FKBP12, FRAP and cyclophilin. In a preferred embodiment, interactors A and B are represented by mutated fragments of FKBP12 and FRAP (FRB T2098L, ARIAD Pharmaceuticals), and the interaction stimulus for this interactor pair is by rapalog AP21967 (ARIAD Pharmaceuticals). Be represented.

別の態様において、ステロイドホルモン レセプタ（例えば、これに限定されないが、エストロゲンレセプタ）のリガンド結合ドメインが、インタラクターＡおよびＢの双方として使用される。係るリガンド結合ドメインは、その同族のホルモンリガンド（このケースではエストロゲン）の添加に際して、ホモ二量体化する（図２２を参照されたい）。 Examples of suitable combinations of interacting protein A and interacting protein B and interacting stimuli are described below:

In another embodiment, the ligand binding domain of a steroid hormone receptor (eg, but not limited to an estrogen receptor) is used as both interactors A and B. Such a ligand binding domain homodimerizes upon addition of its cognate hormone ligand (in this case, estrogen) (see FIG. 22).

  In another embodiment, the full-length or ligand binding domain of a steroid hormone receptor is selected as interactor A, and interactor B is a family of steroid hormone receptor coactivators (SRC-1, GRIP-1, ACTR). A cognate hormone is selected as the interaction stimulus, as described above, selected from among, but not limited to, AIB-1.

One major advantage of the system is the ability to measure protein-protein interactions as simple changes in light intensity. Accordingly, one aspect of the invention relates to a method for detecting protein-protein interactions, the method comprising the following steps:
(A) providing a cell comprising at least:
A first conjugate comprising said first protein of interest and a first terminal complementation protein; and
A second conjugate comprising said second protein of interest and said second terminal complementary protein;
Here, the first conjugate has a preferential position at a cell position that is separate and spatially separated from a position where the second conjugate is preferentially located,
(B) determining whether complementation partner A and complementation partner B are complementary;
Complementarity between the first end and the second end complementary protein is as follows: the first protein of interest is the preferential position of the first protein of interest; It is a method that is an indicator of translocation to the location of the cell that is located and that the two proteins of interest interact (FIG. 21).

  In a typical use of the present method for detecting protein-protein interactions, a stimulus known to cause translocation of the first conjugate is applied prior to step (b). This stimulus may be stress, light, a compound or other means. Also, the compound to be tested is added (but before adding the translocation stimulus). If the compound has the ability to block complementarity, block the interaction between the two proteins of interest, block complementation as such, or translocation of the first conjugate It is possible to prevent

  An example of this method is to determine the interaction (dimerization) between receptors by having the first protein as one receptor and the second protein as another receptor ( Optionally, the same receptor: homodimerization).

  As described above, in a typical configuration of such an assay, the second conjugate further comprises an anchor protein (as described in detail above), the anchor protein being preferentially directed to the first protein of interest. Anchor to a cell location different from the location.

Another advantage of the present invention is that the measured translocation can be a change in light intensity. Accordingly, one aspect of the invention relates to a method for detecting protein translocation, the method comprising the following steps:
(A) providing a cell comprising at least:
A first conjugate comprising said protein, a first end of a complementary protein, and an interaction partner A; and
A second conjugate comprising an anchor protein, a second end of said complementary protein, and an interaction partner B;
Wherein the first conjugate has a preferential position at a cell position that is separate and spatially separated from the position where the second conjugate is preferentially located; and
Wherein said interaction partner A and interaction partner B bind to each other;
(B) determining whether the ends of the complementary proteins are reconstituted;
An indication that the rearrangement (complementarity) between the two ends is the following, ie that the protein is translocated from a preferential position of the protein to a cell position to which the anchor protein anchors. This is the method (FIG. 21).

In a typical use of the present method for detecting protein translocation, a stimulus known to produce translocation of the first conjugate is applied prior to step (b). This stimulus may be stress, light, a compound or other means. Also, the compound to be tested is added (but before adding the translocation stimulus). If this compound has the ability to block complementarity, block the interaction between two interaction partners, block complementation as such, or translocation of the first conjugate It is possible to prevent
As shown in the examples, some spontaneous fluorescence appears. It has now been hypothesized that this is due to the equilibrium of one or both conjugates between various cell locations. Thus, interactions and resulting complementarity occur when they occupy the same space as other interaction partners. This constitutes a sink effect (as described in detail above) since the complementary proteins do not dissociate. This effect may be rather pronounced depending on the system in question. Natural shutting of any naturally translocating protein exposes the anchored interaction partner construct to a constant supply of binding partners, thereby clearly irreversibly binding and prematurely The problem that fluorophore formation occurs is considered.

  One way to solve this problem is to replace the binding interaction partner with an interaction partner (often referred to as a bridging molecule) that requires an interaction stimulus to interact. Such a system provides, for example, a convenient means of allowing the nuclear fraction of the translocating protein to interact with the anchor protein at any particular time.

An example of such an interaction pair is the FKBP12: FRB system (described in detail above). Accordingly, one aspect of the invention relates to a method for detecting protein translocation, the method comprising the following steps:
(A) providing a cell comprising at least:
A first conjugate comprising said protein, a first end of a complementary protein, and an interaction partner A; and
A second conjugate comprising an anchor protein, a second end of said complementary protein, and an interaction partner B;
Here, the first conjugate has a preferential position at a cell position that is separate and spatially separated from a position where the second conjugate is preferentially located,
Wherein said interaction partner A and interaction partner B bind to each other only when an interaction stimulus is applied;
(B) adding an interaction stimulus;
(C) determining whether the first and second ends of the complementary proteins are complementary;
The complementarity between the two ends is as follows: the protein is translocated from a preferential position of the protein to a cell position where the anchor protein anchors after the addition of an interaction stimulus. This is a method (FIG. 21) which is an index of.

In a typical use of the present method for detecting protein translocation, a stimulus known to cause translocation of the first conjugate is applied prior to step (b). This stimulus may be stress, light, a compound or other means. Also, the compound to be tested is added (but before adding the translocation stimulus). If this compound has the ability to block complementarity, block the interaction between two interaction partners, block complementation as such, or translocation of the first conjugate It is possible to prevent
As shown in Example 11, no spontaneous interaction is observed when the FRAP system is used (even if the two conjugates were present in the cytoplasm). As discussed above, the effort in keeping the protein apart does not require more than the natural balance between protein positions is dictated.

Occasionally, the measurement of translocation itself can actually be interesting. However, typically translocation is the result of an underlying cellular process. Some of these underlying processes can be used to obtain information about other processes. For example, when a cell experiences apoptosis, the activity of the caspase enzyme increases. If the peptide sequence (consensus cleavage site for caspase) is cleaved, the method and materials of the present invention allow detection (see FIG. 22). A method for determining apoptosis / caspase activity in a cell comprising at least the following steps:
(A) providing a cell comprising at least:
A first conjugate comprising a first end of a complementary protein, an interaction partner A, a nuclear localization sequence (NLS), a valine-alanine-aspartate (VAD) sequence and an anchor protein;
A second conjugate comprising a second end of a complementary protein, an interaction partner B and an anchor protein;
Wherein the anchor protein of the first conjugate is present at a different cell location than the nucleus, and the anchor protein of the second conjugate is present in the nucleus, and
Here, the first ends of the interaction partner A, the NLS and the complementary protein are all located on the same side of the VAD sequence, and the anchor protein is located on the other side of the NLS. ing;
(B) determining whether the complementary proteins are complementary;
A method in which complementarity is an indication of the following: caspase cleaves the first conjugate, the free part translocates to the nucleus, and partner A and complementation partner B complement;
A kit for detecting an antigen comprising:
(A) a first antibody that binds to the antigen;
(B) a first conjugate comprising a protein that binds to the first antibody and the N-terminus of a complementary protein;
(C) a second conjugate comprising a protein that binds to the first antibody and the C-terminus of the complementary protein;
A kit wherein binding of the first and second conjugates to the first antibody brings the two ends of the complementary proteins in proximity such that a functional protein is formed.

  The thoughts on how to construct and test probes useful in the present invention are described below using (as a specific example) a protein that complements GFP. As will be apparent to those skilled in the art, similar ideas apply to the use of other complementary proteins and their complementary fragments.

  In general, probes (ie, “GeneX” -GFP fusions or GFP “GeneX” fusions) are performed using a “GeneX” specific primer for PCR followed by a cloning step to frame “GeneX”. It is constructed by fusing with GFP. This fusion may contain a specific short vector-derived sequence (eg, multiple cloning sites in the plasmid) between “GeneX” and GFP, and the peptide between “GeneX” and GFP in the fusion protein produced. This results in a linker.

Step-by-step details:
[Identification of gene sequence] This is very easily carried out by searching a genetic information storage organization. Examples of such a storage organization include the GenBank sequence database, which is widely available. Used routinely by molecular biologists. In the specific examples below, the GenBank accession number of the gene in question is provided.

  [Design of gene-specific primer] By checking the gene sequence, it is possible to design a gene-specific primer used in the PCR reaction. Typically, the top strand primer comprises the ATG start codon of the gene and the following ca. If it contains 20 nucleotides 20 but the gene is fused after GFP (ie, a GFP- “GeneX” fusion), the bottom strand primer will contain a stop codon and ca. 20 preceding nucleotides. If the gene is fused before GFP (ie, a “GeneX” -GFP fusion), the stop codon must be removed.

  Optionally, the full-length sequence of GeneX may not be used for the fusion, but may simply be a portion that is localized and redistributed like GeneX in response to a signal.

  In addition to the gene specific sequence, the primer contains at least one recognition sequence for a restriction enzyme, allowing subsequent cloning of the PCR product. The sites are selected so that they are unique in the PCR product and compatible with the cloning vector. Furthermore, an exact number of nucleotides between the restriction enzyme site and the gene specific sequence may be provided to achieve an accurate reading frame and / or translation initiation consensus sequence of the fusion gene. It will be necessary. Finally, the primer always has a small number of nucleotides in front of the restriction enzyme site to allow efficient digestion with the enzyme.

  Identification of the source of the gene to be amplified For a PCR reaction that produces a specific product (with gene-specific primers), the gene sequence must first be present in the reaction (eg, cDNA In the form). GenBank information or scientific literature indicates which tissues the gene is expressed in, and cDNA libraries from very diverse tissues or cell types of various species are commercially available (eg, From Palo Alto, La Jolla and Invitrogen (San Diego)). Many genes are also available from the American Type Tissue Culture Collection (Virginia) in cloned form.

PCR reaction optimization Several factors are known to affect the efficiency and specificity of the PCR reaction, including the annealing temperature of the primer, the concentration of ions present in the reaction (especially Mg ²⁺ and K ⁺ ), As well as the pH of the reaction. If the results of the PCR reaction are unsatisfactory, the parameters described above may not be optimal. Various annealing temperatures should be tested (eg, available from La Jolla etc. in PCR machines with integrated temperature gradients) and / or various buffer compositions should be tried. There are (e.g. OptiPrim buffer system, from Stra Jolla).

  PCR Product Cloning Vectors (the gene product amplified in the vector is cloned and fused to GFP) will already be considered when the primers are designed. When selecting a vector, the practitioner should at least consider which cell type the probe will be subsequently expressed in such a way that the probe's promoter-controlled expression is compatible with the cell. It is. Most expression vectors also contain one or more selectable markers (eg, confer resistance to drugs), which is a useful property when practitioners produce stable transfectants. . The selectable marker should also be compatible with the cells used.

  The actual cloning of the PCR product should not be difficult because it can typically be performed in a one-step cloning of a fragment digested with two different restriction enzymes into a vector digested with the same two enzymes. It is. If it turns out that there is a problem with cloning, it seems that the restriction enzyme did not work well on the PCR fragment. In this case, the practitioner could add longer extensions to the primer end to overcome the potential difficulty of digestion close to the fragment end, or the practitioner would be able to overcome the intermediate cloning step. Could be introduced (not based on restriction enzyme digestion). Several companies provide systems for this approach [eg, San Diego and Palo Alto].

  If the gene is cloned and fused with the GFP gene during this process, the resulting product (usually a plasmid) should be checked to see if it is as expected. The most accurate test would be to obtain the nucleotide sequence of the fusion gene.

Probe testing
Once a DNA construct has been created for a particular probe, its functionality and utility can be tested. In a typical scenario, the two constructs are individually confirmed using full length GFP by subjecting it to the following test: transfecting it into cells that have the ability to express the probe. The cell fluorescence is inspected immediately (typically the next day). In this regard, two characteristics of cellular fluorescence are noted: intensity and sub-cellular localization.

  Its strength should be at least as strong as that of unfused GFP, usually in cells. Otherwise, the sequence or quality of the probe DNA appears to be incorrect and should be checked carefully.

  Subcellular location is an indication of whether the probe can work well.

  If it localizes as expected for the gene in question (eg it is excluded from the nucleus), it is immediately transferred to a functional test. If the probe is not localized immediately after the transfection procedure, it may be due to overexpression in time at this point, since cells are generally very Because many copies of the plasmid are obtained and localization occurs in time (eg, within a few weeks), plasmid copy number and expression levels decrease accordingly.

  If localization does not occur after a prolonged time, the localization function is disrupted by fusion with GFP (eg, a protein that is essential for interaction with its normal cellular anchor protein) Masking the array). In this case, the reverse fusion may function (eg, if GeneX-GFP does not function, GFP-GeneX may function), because two different parts of GeneX are in close proximity to GFP. This is because it is influenced by proximity. If this does not work, the proximity of GFP to either end seems to be a problem and attempts to increase the distance by introducing a longer linker between GeneX and GFP in the DNA construct. Will be able to.

  If there is no prior knowledge of localization and no localization is observed, it may be because the probe should not be localized at this point, because such events are predispositions for proteins fused to GFP Because it depends on. It should then be tested for functional testing.

  In a functional test, a cell expressing a probe is treated with at least one compound, which disrupts the signal transduction pathway (usually by activation), in which the probe is It is expected to be presented by redistributing itself within the cell. If the redistribution is as expected, for example, if preliminary knowledge indicates that it translocates from position X to position Y, then pass the first critical test. In this case, it proceeds further to response characterization and quantification.

  If it does not function as expected, it may be due to the cell being deficient in at least one component of a signal transduction pathway (eg, a cell surface receptor) or having a species incompatibility (eg, If the probe is designed based on the sequence information of the human gene product and the cell is of hamster origin). In both examples, the practitioner should identify other cell types for the process to be tested, where potential problems will not apply.

  If this is the case, the conjugate is constructed with fragments of the complementary protein instead of the full length and tested when expressed in the same cell. If the redistribution characteristics cannot be identified, the problem may be as described above and the probe should be retested under more optimal cellular conditions.

  If the probe does not function under optimal cellular conditions, start over.

  Many existing cellular systems for transfection. A few examples of mammalian cells are directly from healthy or diseased animals (primary cells), or transfected mammalian cells (cell lines) that have the ability to replicate indefinitely under cell culture conditions. Isolated. The term “mammalian cell” means any living cell of mammalian origin. The cells may be established cell lines, many of which are available from the American Type Tissue Culture Collection (ATCC, Virginia, USA) or similar culture collections (Culture Collections). The cell has a limited life span and is a newly established primary cell derived from a mammalian tissue, including a tissue derived from a transgenic animal, or a primary cell derived from a mammalian tissue (including a transgenic tissue). A hybrid cell or cell line (eg, a hybridoma cell line) derived from fusing different cell types from mammals. The cells may include one or more non-native gene products (e.g., receptors, enzymes, enzyme substrates) in addition to or prior to the fluorescent probe (in addition to). It may be expressed optionally. Suitable cell lines include (but are not limited to) the following: fibroblast origin (eg, BHK, CHO, BALB, NIH-3T3) or endothelial origin [eg, HUVEC, BAE (bovine arterial endothelium), CPAE (cow pulmonary artery endothelium), HLMVCEC (human pulmonary microvascular endothelial cells)], or of respiratory epithelial origin (eg, BEAS-2B), or of pancreatic origin ( For example, RIN, INS-1, MIN6, bTC3, aTC6, bTC6, HIT) or of hematopoietic origin [eg primary cells isolated from humans, including monosites, macrophages, neutrophils, basophils Spheres, eosinophils and lymphocyte populations (AML-14, AML-193, HL-60, RBL-1, U937, RAW, JAWS), Is of adipocyte origin (eg 3T3-L1, human preadipocytes), or of neuroendocrine origin (eg AtT20, PC12, GH3), muscle origin (eg SKMC, A10, C2C12), kidney origin (Eg, HEK293, LLC-PK1) or those of neuronal origin [eg, SK-N-DZ, SK-N-BE (2), HCN-1A, NT2 / D1].

  Examples of the present invention are based on CHO cells. Accordingly, cell lines derived from fibroblasts (eg, BALB, NIH-3T3 and BHK cells) are preferred.

  It is preferred to introduce the heterologous conjugate as a plasmid into the cell, eg, by mixing individual plasmids with a suitable transfection agent (eg, FuGENE) when applied to the cell, Simultaneously express and integrate all heterologous conjugates (or GFP fragments). The plasmids encoding each conjugate have different genetic resistance markers, allowing selection of cells expressing those conjugates: and each conjugate is distinct Preferably it comprises an amino acid sequence (eg HA or myc or Flag marker), which can be detected immunocytochemically so that the expression of these conjugates in the cells can be easily confirmed. Many other means for introducing one or both conjugates can be performed equally, such as electroporation, calcium phosphate precipitation, microinjection, adenoviral and retroviral methods, conjugates of both For example, a bicistronic plasmid encoding.

  In the present invention, the term “protein” is a term that should have a general meaning.

  This includes not only translated proteins, peptides or protein fragments, but also chemically synthesized proteins. It is expected that natural or induced post-translational modifications (eg, glycosylation and lipidation) will occur on proteins translated in the cell, and their products are still proteins. it is conceivable that.

  The term “intracellular protein interaction” generally refers to an interaction between two proteins within the same cell, as described above. Said interactions are covalent and between protein components, most commonly between one or more regions or domains in each protein (these physicochemical properties allow some specific recognition) and Depending on non-covalent forces and the subsequent interaction between the two protein components involved. In a preferred embodiment, the intracellular interaction is a protein-protein interaction.

Fluorescence recording varies depending on the problem of the method in question. In one aspect, the emitted light is measured with various devices known to those skilled in the art. Typically, such a device comprises the following components: (a) a light source;
(B) a method for selecting the wavelength of light from the light source (exciting the emission of the luminophore);
(C) an apparatus for quickly blocking or passing inflow of excitation light into the rest of the system;
(D) a series of optical elements to transmit excitation light to the analyte, collect the emitted fluorescence in a spatially resolved manner, and form an image from this fluorescence radiation (or Another type of intensity map related to the method of detection and measurement),
(E) a bench or stand holding a container of cells, measured with a predetermined geometry for a series of optical elements;
(F) a detector for recording the light intensity (preferably in the form of an image);
(G) a computer or electrical system and associated for acquiring and storing recorded information and / or images, and for calculating the degree of redistribution from recorded images software.

  In a preferred aspect of the present invention, the device system is automated. In one aspect, the components in (d) and (e) above include a fluorescence microscope. In one aspect, the component in (f) above is a CCD camera.

  In one aspect, the component in (f) above is an array of photomultiplier tubes / devices.

  In one embodiment of the present invention, the actual fluorescence measurement is made in a standard type of fluorometer (fluorescence plate reader) for microtiter type plates.

  In one embodiment, using an optical scanning system, the bottom surface of the microtiter plate is illuminated as follows. That is, irradiation is performed so that time-resolved recording of changes in luminescence or fluorescence can be obtained simultaneously from all spatial limitations.

  In one embodiment, the image is formed and recorded by an optical scanning system.

  There are various instruments for measuring light intensity. In one embodiment, a fluorescent plate reader is used (eg, Wallac Victor (BD Biosciences), Spectrafluor (Tecan), Flex station (Molecular Devices), Explorer (Accumen)). In another embodiment, an imaging plate reader is used [eg, FLIPR (Molecular Devices), LeadSeaker (Amersham), VIPR (Molecular Devices)]. In another embodiment, an automated imager is used (such as Arrayscan (Cellomics), Incell Analyzer (Amersham), Opera (Evotec)). In still further embodiments, a confocal fluorescence microscope is used (eg, LSM510 (Zeiss)).

  One particular advantage of this method is that it can be performed on heterogeneous cell populations. This avoids, inter alia, the steps necessary to obtain clonal cells.

  This is achieved by fluorescence activated cell sorting (FACS) prior to testing. One step in the process is the removal of the most green cells, which are cells where functional fluorescence is achieved (even if the two proteins of interest are not assumed to interact). ). Another step is the removal of melanocytes, which are cells in which the two heterologous conjugates do not interact (eg, functional complementarity does not occur or slightly occurs). This event may be due to lack of transfection in these cells, poor expression ratio between the two constructs, or lack of functional expression of either construct. It is currently anticipated that transfection does not occur as desired in both the green and black cells, and there is no, poor, or excess heterogeneous conjugate complementation. The resulting “moderate to low green” cells are then used in any of the above methods or other complementation-based methods. The “most green”, “moderately green”, “lowly green” and “black” cells, respectively, have reduced levels of fluorescence compared to each other. These levels are predetermined by those skilled in the art for relative production. Suitable methods for detecting interactions between proteins of interest additionally include FACS. The purpose of this second FACS process is to separate cells with a large dynamic range. The first action in additional FACS is to stimulate “moderate to low green” FACS cells with a compound (which induces an interaction between the two proteins of interest). Yes, and then allow sufficient time to allow the proteins to interact and fluorescent protein fragments to fold and emit fluorescence (become fluorescent). The next action is to subject them to a second FACS to remove dark cells and moderate green cells.

  The remaining population of cells (the most green cells or very green cells (VGC)) has a low to moderate background, and upon interaction between the two proteins of interest, fluorescent proteins Still have the ability to form. If the cells grow to a sufficient number and the fluorescence is diluted after many generations, the cells can be used immediately in any of the methods outlined above (eg, the interaction between the two proteins of interest Detecting compounds that induce action and screening for compounds that interfere with the conditional interaction between the two protein components).

  In a preferred aspect of the method, the at least one cell is a mammalian cell.

  The term “compound” means any sample, which has a biological function or exerts a biological effect in a cellular system. The sample may be a sample of biological material, for example a sample of body fluid (including blood, plasma, saliva, milk, urine), or a microbial or plant extract, contaminants An environmental sample containing (including heavy metals or poisons), or a sample containing a compound or mixture of compounds prepared by organic synthesis or genetic techniques Also good. The compounds may be small organic compounds or biopolymers, including proteins and peptides.

  In another preferred aspect of the method, the heterologous conjugate is a fusion protein.

文献： Ｈｅｉｍ， Ｒ．， Ｐｒａｓｈｅｒ， Ｄ． Ｃ．， ａｎｄ Ｔｓｉｅｎ， Ｒ． Ｙ． （１９９４） Ｗａｖｅｌｅｎｇｔｈ ｍｕｔａｔｉｏｎｓ ａｎｄ ｐｏｓｔｔｒａｎｓｌａｔｉｏｎａｌ ａｕｔｏｘｉｄａｔｉｏｎ ｏｆ ｇｒｅｅｎ ｆｌｕｏｒｅｓｃｅｎｔ ｐｒｏｔｅｉｎ． Ｐｒｏｃ． Ｎａｔｌ． Ａｃａｄ． Ｓｃｉ． Ｕ．Ｓ．Ａ． ９１，１２５０１−１２５０４。 One aspect of the present invention relates to multiplexed splitting fluorophore complementation using different colors. Where two potentially fluorescent complexes have distinct fluorescent excitation or emission properties or both, ligating one fluorescent protein fragment with two or more appropriate complementary fragments ( By combining, it is possible to determine the degree of binding between the first fluorescent protein fragment and either of the two other complementary fragments. Typically, the first fluorescent protein fragment is fused to a specific protein, which is capable of binding to any of the three other proteins, each of which is suitably combined with a separate complementary fragment. It is fused. For example, the first fluorescent protein fragment may be a C-terminal fragment of enhanced GFP (EGFP, SEQ ID NO: 1) obtained by splitting EGFP after residue 80. The three complementary fragments may be the appropriate N-terminal fragments of EGFP, EGFP Y66W (SEQ ID NO: 2) and EGFP Y66H (SEQ ID NO: 3). The three EGFP variants have different spectral characteristics:

Literature: Heim, R.D. Prasher, D .; C. , And Tsien, R .; Y. (1994) Wavelength mutations and posttranslational autooxidation of green fluoresce protein. Proc. Natl. Acad. Sci. U. S. A. 91, 12501-12504.

  For example, a fluorescent complex produced by constructing the C-terminal half of GFP (eg, residues 158 to 238) and the corresponding N-terminal half of GFP, GFP Y66W or GFP Y66H (eg, residues 1 to 157) The body will have distinctly distinct fluorescent properties. Also, the relative amount of complex in the mixture can be calculated.

  Not only can various colors be used as illustrated above, but other physical parameters of the fluorophore can also be altered (eg, intensity, fluorescence lifetime, folding time, etc.).

  Two-color multiplexed partitioning involves several uses: In many cases (as described above), the protein of interest is linked to the “constant” half of the fluorophore, while its mutual Each of the working partners is linked to a “variable” portion of the fluorophore (eg, those that upon fusion yield a green fluorophore and a blue fluorophore). This gives spatial information (ie that two different interaction partners are in different positions) by indicating where the protein of interest is located by color. Also, interaction partners can be in the same location, where the color gives an indication of which interaction partner your protein will prioritize at any particular time. Finally, as a special case of the latter, the interaction of your protein with different partners is regulated, for example by post-translational modification, and the color indicates whether your protein has been modified or not. These three different setups, in the broadest possible sense, can be used as sensors for the physical state of your protein, or they can be used in screening assays (in this assay, You measure the ability to change the ratio between the two color readouts of the test compound).

  Finally, color is the only physical parameter of GFPs. Other physical parameters (these can correspond to specific amino acids in the GFP sequence and these are easily detectable, such as absorption spectra, fluorescence lifetime, time for maturation of fluorophore, etc.) Can be used in exactly the same manner as the color. The number of different interaction partners need not be limited to two.

  One special example shows how the method can be used as a tool for using spatial information about screening. This is true even if there is no known interaction partner for a particular protein at different cell locations (or even if it does not have any interaction partner in one or more of those areas). ) Can be used. Cystic fibrosis is probably the most frequently studied protein transport disease. Cystic fibrosis Transmembrane conductance regulator (CFTR) is a multi-membrane spanning protein (usually as a Cl efflux channel in the plasma membrane at the tip of epithelial cells of the respiratory tract) Function). The most common mutation (Delta F508) retains the protein in the endoplasmic reticulum (ER) and reduces the amount of CFTR expressed in the plasma membrane of epithelial cells, thereby reducing Cl efflux from the cell. This Delta F508 ER retention defect is reversible and induces Delta F508 to the plasma membrane and increases Cl permeability by reducing temperature, several small molecules, and induction of “chaperones” However, many studies have revealed it. One method of screening for compounds that modify the behavior of mutant CFTR uses the concept of a segmented fluorophore / multiple colors. Mutant CFTR can be expressed intracellularly as a fusion with a zipper fragment (fused to the “constant” half of the fluorophore). Expression in the same cell fusion of ER and plasma membrane markers and zippers (fused with different colors) indicates that this produces a color when the CFTR variant reaches the ER. When the CFTR moves to the PM (eg, due to a drug stimulus), this generates a different color. Thus, screening for compounds that facilitate the production of “PM color” can find molecules that specifically correct ER retention defects in CF patients.

One aspect of the invention therefore relates to a method for producing a library of proteins that interact in living cells, said method comprising:
1. Introducing two sets of plasmids simultaneously or sequentially into a pool of cells (one set of plasmids encoding a library of protein A, each fused to the N-terminal half of a complementary fluorophore; And a second set of plasmids encodes a library of protein B, each fused to the C-terminal half of the complementary fluorophore).
2. Where the functional fluorophore is formed at the correct location by imaging the cell and where the functional fluorophore is not formed or where a functional fluorophore is formed but formed in the wrong cell compartment (Formation of the functional fluorophore is an indication that the interaction occurs between protein A and B in the correct cell compartment).

  For example, for proteins that move from one cell location to another in response to a stimulus, the binding partner at each location can be identified.

  In one aspect of the invention, the method results in a disruption of the bond between two proteins when located in one cell compartment or location but not in another cell compartment or location. It relates to drug identification. This aspect is basically executed as described above, but differs only in the following points. The difference is that instead of sorting the cells based on intensity, the cells are imaged with a standard imaging facility to determine not only that binding will occur, but also where such binding will occur. It is.

  This system is also useful for screening for fluorophores with novel properties, such as those that can be used in the utilization of the above-described split fluorophore complementation.

  By mutating both the N- and C-termini of GFP in this system, the system is used to select double (or more) fluorophore variants with novel properties in a combinatorial manner. This provides a wider selection for selecting objects. Furthermore, since the two variants are present in different halves of the molecule, they can be additive or compensatory or both. Finally, the fact that they were discovered using a split fluorophore complementation system immediately means that they can be used as sensors in the system.

  The invention is further illustrated in the following non-limiting examples.

アライメントは図１６に記載される。 Example 1: Fluorescent protein alignment

The alignment is described in FIG.

Example 2: Construction of an EGFP complementary fragment probe
Antiparallel leucine zippers (referred to as NZ and CZ), which can bind to each other in prokaryotic and eukaryotic, were fused to different fragments of GFP to evaluate the optimal site for splitting GFP (of such fragments). (For use in molecular complementation experiments, including fluorescence reconstruction experiments). Annealing and ligating NZ and CZ leucine zippers with phosphorylated oligonucleotides 2110-2115 (for NZ zippers, see Table 2) or phosphorylated oligonucleotides 2116-2121 (for CZ zippers) Were prepared in the NcoI-BamHI cut pTrcHis-A vector (available from Invitrogen) production vector PS1515 (expression vector encoding NZ zipper) or PS1516 (expression vector encoding CZ zipper). The oligos ligated to NZ and CZ annealing mixture 1 produced coding sequences for the N-terminal part of NZ and CZ zippers. The oligos ligated to NZ and CZ annealing mixture 2 produced the coding sequence of the middle part of NZ and CZ zippers, and the oligos ligated to NZ and CZ annealing mixture 3 produced NZ and CZ zippers. A coding sequence for the C-terminal portion of was produced.

Annealing primer pair for NZ zipper
NZ annealing mixture 1
Forward oligo 2110 (1 μM) 5 μl
Reverse oligo 2111 (1 μM) 5 μl
50 mM Tris-HCI, 10 mM MgCl ₂ , pH 8.0 2 μl
8 μl of H ₂ O

NZ annealing mixture 2
Forward oligo 2112 (1 μM) 5 μl
Reverse oligo 2113 (1 μM) 5 μl
50 mM Tris-HCl, 10 mM MgCl ₂ , pH 8.0 2 μl
8 μl of H ₂ O

NZ annealing mixture 3
Forward oligo 2114 (1 μM) 5 μl
Reverse oligo 2115 (1 μM) 5 μl
50 mM Tris-HCl, 10 mM MgCl ₂ , pH 8.0 2 μl
8 μl of H ₂ O

  Each annealing mixture is heated on a preheated Hybaid OmniGene PCR machine at 80 ° C. for 2 minutes, subsequently stopped, and cooled at room temperature (approximately 10 minutes). The pieces were subsequently placed on ice.

Annealing primer pair for CZ zipper
CZ annealing mixture 1
Forward oligo 2116 (1 μM) 5 μl
Reverse oligo 2117 (1 μM) 5 μl
50 mM Tris-HCl, 10 mM MgCl ₂ , pH 8.0 2 μl
8 μl of H ₂ O

CZ annealing mixture 2
Forward oligo 2118 (1 μM) 5 μl
Reverse oligo 2119 (1 μM) 5 μl
50 mM Tris-HCl, 10 mM MgCl ₂ , pH 8.0 2 μl
8 μl of H ₂ O

CZ annealing mixture 3
Forward oligo 2120 (1 μM) 5 μl
Reverse oligo 2121 (1 μM) 5 μl
50 mM Tris-HCl, 10 mM MgCl ₂ , pH 8.0 2 μl
8 μl of H ₂ O

  Each annealing mixture is heated on a preheated Hybaid OmniGene PCR machine at 80 ° C. for 2 minutes, subsequently stopped, and cooled at room temperature (approximately 10 minutes). The pieces were subsequently placed on ice.

Restriction enzyme digestion of pTrcHis-A prokaryotic expression vector
The prepared NZ and CZ leucine zipper coding sequences were cloned using the pTrcHis-A prokaryotic expression vector (cut with NcoI and BamHI restriction enzymes and gel purified):

Restriction enzyme digestion of pTrcHis-A vector
pTrcHis-A (1 μg / μl) 2 μl
NcoI (10 U / μl) 1 μl
Nhel (5U / μl), optional 0.5μl
BamHI (20 U / μl) 1 μl
100xBSA 0.4μl
10 × NEB (New England Biolabs, NEB) BamHI buffer
3 μl
23 μl of H ₂ O
Calf intestinal phosphatase (optional, last 20 min only) 0.5 μl

  The vector was digested for about 1 hour at 37 ° C. and purified by agarose gel electrophoresis. The desired vector fragment was recovered from the gel using Qiagen's QIAquick gel extraction kit (spin column) and recovered in 50 μl elution buffer. Nhel (which cuts between NcoI and BamHI) was included to minimize the amount of uncleaved and self-ligated vector.

Ligation and transformation of annealed NZ oligo pairs
Each of the three NZ annealing mixtures 1-3 was diluted 50-fold (1 μl of the mixture into 50 μl H ₂ O), mixed and ligated to the cleaved vector as follows (20-24 ° C. for 3 hours) ):

Ligation of NZ zipper fragment to pTrcHis-A vector
1 μl of annealing mixture 1
Annealing mixture 2 1 μl
Annealing mixture 3 1 μl
10xT4 DNA ligase buffer (New England Biolabs)
1 μl
T4 DNA ligase (400 U / μl, New England Biolabs)
0.5 μl
pTrcHis-A (NcoI + BamHI cleavage) 0.5 μl
5 μl of H ₂ O

  Alternatively, the fragments in the NZ annealing mixture 1, 2, and 3 can be ligated in the absence of the vector and purified by agarose gel electrophoresis (before ligation to the NcoI-BamHI cut vector). The annealed and ligated oligonucleotides from annealing mixtures 1-3 had a single-stranded terminal overhang, which was compatible with the overhang formed by NcoI and BamHI restriction enzyme digests of pTrcHis-A. It was a thing. After ligation of the fragment to cleaved pTrcHis-A, the NcoI and BamHI sites were regenerated.

  Following ligation to the vector, 2 μl of the ligation mixture was transformed into 50 μl of One Shot TOP10 chemically competent E. coli cells (Invitrogen) (according to manufacturer's protocol). Ligation can be performed using different amounts or volumes of fragments and buffers. The inserted DNA sequence (SEQ ID NO: 7) and the encoded NZ zipper peptide (SEQ ID NO: 8) are as follows:

  The Gly-Gly-Thr-Gly-Ser-Gly amino acid sequence at the end is part of the linker sequence, which was inserted between the NZ zipper peptide and the N-terminal fragment of EGFP (NtermEGFP). In addition, the zipper sequence in the NtermEGFP-NZ fusion protein is also Gly-Gly-Thr-Gly-Ser-Gly, and the Gly-Gly-Thr-Gly coding sequence is represented by NtermEGFP reverse amplification primers 2129, 2130, and 2131 (Table 3). It has been repeated. Underlined are the unique NcoI (CCATGG), Agel (ACCGGT) and BamHI (GGATCC) sites, which clone the zipper peptide to pTrcHis-A and the NtermEGFP-NZ fragment to the NZ zipper vector PS1515 (see below) Used for. An asterisk (*) indicates a stop codon.

Ligation and transformation of annealed CZ oligo pairs
Each of the three CZ annealing mixtures 4-6 was diluted 50-fold (1 μl of the mixture into 50 μl of H ₂ O) and mixed as follows:

Ligation of CZ zipper fragment to pTrcHis-A vector
CZ annealing mixture 1 1 μl
CZ annealing mixture 2 1 μl
CZ annealing mixture 3 1 μl
10xT4 DNA ligase buffer (New England Biolabs)
1 μl
T4 DNA ligase (400 U / μl, New England Biolabs)
0.5 μl
pTrcHis-A (NcoI + BamHI cleavage) 0.5 μl
5 μl of H ₂ O

  Alternatively, the fragments in the CZ annealing mixtures 1, 2, and 3 can be ligated in the absence of the vector and purified by agarose gel electrophoresis (before ligation to the NcoI-BamHI cut vector). The annealed and ligated oligonucleotides from annealing mixtures 1 to 3 had a single-stranded terminal overhang that was compatible with the overhang produced by NcoI and BamHI restriction enzyme digestion of pTrcHis-A. It was sex. After ligation of the fragment to cleaved pTrcHis-A, the NcoI and BamHI sites were reshaped.

  Following ligation to the vector, 2 μl of the ligation mixture was transformed into 50 μl of One Shot TOP10 chemically competent E. coli cells (Invitrogen) (according to manufacturer's protocol). Ligation can be performed using different amounts or volumes of fragments and buffers. The inserted DNA sequence (SEQ ID NO: 9) and the encoded CZ zipper peptide (SEQ ID NO: 10) are as follows:

  The Gly-Gly-Thr-Gly amino acid sequence at the end is part of the linker sequence, which was inserted between the CZ zipper peptide and the C-terminal fragment of EGFP (CtermEGFP). The zipper sequence in the CZ-CtermEGFP fusion protein is also Gly-Gly-Thr-Gly, and the Thr-Gly coding sequence is repeated in CtermEGFP forward amplification primers 2133, 2134, and 2135 (Table 3). Underlined are the unique NcoI (CCATGG), Agel (ACCGGT) and BamHI (GGATCC) sites, which clone the zipper peptide to pTrcHis-A and the CZ-CtermEGFP fragment to the CZ zipper vector PS1516 (see below) Used for. An asterisk (*) indicates a stop codon.

Example 3: E. coli colony PCR selection, plasmid miniprep and DNA sequencing
Transformed bacteria were plated on Luria broth (LB) agar plates. The plate contains 100 μl / ml carbenicillin as a selective agent (present in the E. coli medium used). To quickly identify transformants containing the desired NZ or CZ construct, colony PCR screening was performed using oligo 2110 (5 ′ forward NZ oligo) and 2115 (3 ′ reverse NZ oligo) or oligo 2116 ( 5 ′ forward CZ oligo) and 2121 (3 ′ reverse CZ oligo) were used:

Per sample (15 μl reaction volume)
10 × Taq polymerase buffer (Perkin Elmer) 1.5 μl
dNTP (5 mM nucleotide, each) 0.3 μl
50 mM MgCl ₂ 0.6 μl
Dimethyl sulfoxide (DMSO) 0.3 μl
Taq polymerase (Perkin Elmer) 0.2 μl
5 ′ forward primer (10 μM) 0.5 μl
3 ′ reverse primer (10 μM) 0.5 μl
6.1 μl of H ₂ O
5.0 μl of transformant resuspended in H ₂ O

Cycling parameters (RoboCycler Gradient 96, Stratagene)
Initial denaturation at 94 ° C. for 3 minutes, followed by 25 cycles of the steps described below (all steps for 1 minute):
Denaturation is performed at 94 ° C, primer annealing at 53 ° C and primer extension at 72 ° C.
Finally, an additional extension step was performed at 72 ° C. (5 minutes).
Sixteen NZ transformants and 16 CZ transformants were selected. PCR fragments with an expected product size of approximately 120 base pairs were amplified from 14 NZ clones and 15 CZ clones (determined by agarose gel electrophoresis analysis).

  Three positive colonies were picked from each transformation (NZ and CZ) and seeded in 5 ml liquid LB medium. After overnight culture at 37 ° C., the plasmid DNA was purified by mini-preparation using the QIAprep kit (Qiagen).

  Plasmids containing the correct NZ (PS1515) or CZ (PS1516) fragment insert were identified by DNA sequencing (using forward sequencing primer 1282 on an ABI prism model 377 DNA sequencer).

Example 4: Prokaryotic expression vector encoding EGFP fragment and zipper fusion protein
The DNA sequences encoding the NZ and CZ zippers in the respective prokaryotic expression vectors PS1515 and PS1516, the desired EGFP fragment (the N-terminal fragment of EGFP is referred to as NtermEGFP, and the C-terminal fragment of EGFP is CtermEGFP) Or a linker sequence at either the 5 ′ end (in NZ vector PS1515) or 3 ′ end (in CZ vector PS1516) of the leucine zipper coding sequence. Fusion can be achieved by using a combination of a properly positioned unique AgeI restriction enzyme recognition site and either the unique NcoI or BamHI site used to clone the zipper-encoded fragment (DNA and amino acid sequences are shown above) That).

  The general structure of the fusion protein coding sequence is shown in FIG.

  For example, a prokaryotic expression vector (the vector encodes a fusion protein, which is fused (N-terminally) via the N-terminus of a NtermEGFP fragment (eg, residue 1-172 (NtermEGFP172)). NZ zipper (ie, fused to the C-terminus of the NtermEGFP fragment), this region of the EGFP coding sequence in the commercial expression vector pEGFP-C1 (Clontech) is Amplification was by PCR using 2128 (containing a unique NcoI site) and reverse oligo 2131 (containing a unique Agel site).

Per sample (50 μl reaction volume)
10 × Pfu polymerase buffer (Stratagene) 5.0 μl
dNTPs (5 mM nucleotides, each) 1.0 μl
Pfu Hot Start Polymerase (Stratagene) 1.0 μl
5 ′ forward primer (10 μM) 1.0 μl
3 ′ reverse primer (10 μM) 1.0 μl
pEGFP-C1 vector (10 ng / μl) 2.0 μl
H ₂ O 39.0 μl

Cycling parameters (Hvbaid OmniGene PCR machine)
Initial denaturation at 94 ° C. for 3 minutes, followed by 25 cycles of the steps described below (all steps for 1 minute):
Denaturation is performed at 94 ° C, primer annealing at 53 ° C and primer extension at 72 ° C.
Finally, an additional extension step was performed at 72 ° C. (5 minutes).

  PCR fragment encoding the desired EGFP fragment (eg, the above fragment composed of residues 1-172) (having a suitably engineered terminal restriction enzyme recognition site included in the primer sequence) ) And then ligated to the constructed NZ or CZ prokaryotic leucine zipper expression vector PS1515 or PS1516 (cut with the same enzymes), cut with NcoI and Agel or Agel and BamHI as described above. Gel purified:

Restriction enzyme digestion of NtermEGFP and CtermEGFP PCR fragments
EGFP fragment (gel purified) 26 μg
NcoI (10 U / μl) or BamHI (20 U / μl) 0.5 μl
Agel (10 U / μl) 1.0 μl
10 × New England Biolabs buffer 2 3 μl

Restriction enzyme digestion of NZ (PS1515) and CZ (PS1516) vectors
Vector (1 μg / μl) 1.0 μl
NcoI (10 U / μl) or BamHI (20 U / μl) 0.33 μl
Agel (10 U / μl) 0.66 μl
1 × l of 10 × New England Biolabs buffer 2
₇ μl of H ₂ O

  All enzymes were obtained from New England Biolabs. The DNA preparation was digested for 1 hour at 37 ° C. and gel purified.

Ligation of EGFP fragment to cleaved PS1515 or PS1516 vector
2 μl of cut and purified vector
Cleaved and purified NtermEGFP or CtermEGFP fragment 4 μl
10xT4 DNA ligase buffer (New England Biolabs)
1 μl
T4 DNA ligase (400 U / μl, New England Biolabs)
0.5 μl
H ₂ O 2.5 μl

  Ligation was allowed to proceed for 30 minutes at 22 ° C., after which 2 μl of each ligation mixture was transformed into 50 μl of One Shot TOP10 chemically competent E. coli cells (Invitrogen) (according to manufacturer's protocol).

  Transformed cells were plated on LB plates (containing carbenicillin) and plasmids were prepared as described above from two colonies of each transformation.

Example 5: EGFP-based reconstitution in E. coli
Plasmids expressing functional NtermEGFP-NZ or CZ-CtermEGFP complementation constructs were used in 10 μl of One Shot TOP10 chemically competent E. coli cells (Invitrogen) each with 1 μl of appropriately matched NtermEGFP-NZ or CZ-CtermEGF Co-transforming a plasmid (ie, a plasmid expressing an EGFP fragment, the plasmid being truncated at the same splitting site (NtermEGFP fragment) or before (CtermEGFP fragment)); And co-transformed cells were treated with LB plates (carbenicillin and 5 mM isopropyl-β-thiogalactosin And be plated on containing (IPTG)), it was identified by.

  Transformed cells were cultured overnight at 37 ° C. E. coli colonies that were illuminated with a blue light source (Fiberoptic-Heim LQ2600) and were confirmed to be green fluorescent due to EGFP-based reconstitution when viewed through yellow filter glasses were transformed (fluorescence was at least 5 days at 5 ° C) Visible on the agar plate without expansion in about 10-20 hours (which occurred further during storage of the plate).

  Functional complementarity was clearly visible in cells co-transformed with complementation constructs based on splitting either between residues 157 and 158 or between residues 172 and 173, and The DNA sequence of the expression vector that produces a functional NtermEGFP-NZ or CZ-CtermEGFP complementary fragment (referred to as PS1594, PS1595, PS1596, PS1597, see Table 4) is the DNA sequence using primer 1282. Validated by Thing (as previously described).

  Surprisingly, the E. coli colonies of cells co-transformed with a vector expressing an EGFP complementary fragment having a split in the lle171-Ser175 loop (ie, between residues 172 and 173, vectors PS1595 and PS1597) are Ala154-Gly160 loops. It was significantly more fluorescent than the colonies of cells co-transformed with a vector expressing an EGFP complementary fragment with a split on (ie, between residues 157 and 158, vectors PS1594 and PS1596).

  Functional complementarity was not clearly visible in cells co-transformed with a complementation construct based on the split between residues 144 and 145. DNA sequencing confirmed that expression vectors PS1614 and PS1615 encoded the correct NtermEGFP-NZ and CZ-CtermEGFP reconstructed fragments, respectively.

Example 6: Eukaryotic expression vector encoding EGFP fragment and zipper fusion protein
Since the fluorescent signal produced by the complementary fragment based on the 144/145 split fragment is low, only the complementary fragment based on the split at residues 157/158 or 172/173 will enter the eukaryotic expression system. And was approved to assess fragment complementation in mammalian cells.

  NtermEGFP-NZ fragment (in PS1596 and PS1597), and CZ-CtermEGFP fragment (in PS1594 and PS1595) have NcoI sites (5 'to start codon) and BamHI sites (3' to start codon) on both sides. It is flunked. The fragment was introduced as a blunt-ended NcoI / BamHI fragment into a mammalian expression vector (cut with Eco47III / BamHI). To select for stable expression of plasmids expressing both NtermEGFP-NZ and CZ-CtermEGFP, the expression vectors for NtermEGFP-NZ and CZ-CtermEGFP fragments were selected against neomycin / geneticin / G418 and zeocin, respectively. Contains a marker.

  Plasmids PS1594, PS1595, PS1596, and PS1597 were cut with NcoI restriction enzyme, blunted with Klenow fragment, gel purified, cut with BamHI, and gel purified (as described below).

Restriction enzyme digestion of NtermEGFP-NZ and CZ-CtermEGFP prokaryotic expression vectors
PS1594, PS1595, PS1596, or PS1597 (1 μg / μl)
1 μl
NcoI (10 U / μl, from New England Biolabs)
1 μl
3x 10x buffer 4 (NEB)
H ₂ O 25 μl

  The plasmid was digested for about 1 hour at 37 ° C. 1 μl of 1 mM dNTP mixture and 1 unit of Klenow fragment (New England Biolabs) were added and the reaction was incubated for 30 minutes at room temperature. The linear plasmid fragment was purified by agarose gel electrophoresis, recovered from the gel using Qiagen's QIAquick gel extraction kit (spin column), and recovered in 50 μl of elution buffer. 5 μl BamHI buffer (New England Biolabs) and 10 units BamHI enzyme were added. The plasmid was digested for about 1 hour at 37 ° C. The desired plasmid fragment was purified by agarose gel electrophoresis and recovered from the gel using Qiagen's QIAquick gel extraction kit (spin column) and recovered in 50 μl elution buffer.

  In order to stably co-express NtermEGFP-NZ and CZ-CtermEGFP fragments in the same mammalian cells, mammalian expression vectors carrying different selectable markers are required. To achieve this, this plasmid was designated PS0609, in which the kanamycin / neomycin selection marker in the expression vector pEGFP-C1 was replaced with a zeocin resistance marker.

Replacement of kanamycin / neomycin marker with zeocin marker in pEGFP-C1
pEGFP-C1 was digested with AvrII (the kanamycin / neomycin selectable marker was excised), then gel purified and the vector fragment was ligated with an approximately 0.5 kbp AvrII fragment (encoding zeocin resistance). This fragment was isolated by PCR amplification of the zeocin selectable marker in plasmid pZeoSV (Invitrogen) using primers 9655 and 9658 (see Table 2). Both primers have an AvrII cloning site and are flanked on both sides of the zeocin resistance gene on the plasmid pZeoSV (which contains its own E. coli promoter). The top primer 9658 contains an AseI site at the start of zeocin, which can be used to determine the orientation of the AvrII insert relative to the SV40 promoter (which exerts resistance in mammalian cells). The resulting plasmid is called PS0609.

  Plasmid pEGFP-C1 (Clontech) and its zeocin resistant derivative PS0609 were cut with Eco47III restriction enzyme, gel purified, BamHI cut and gel purified (as described below). These steps extract EGFP and leave the rest of the vector intact.

Restriction enzyme digestion of eukaryotic expression vectors
pEGFP-C1 or PS0609 DNA (1 μg / μl) 0.5 μl
Eco47III (10 U / μl, from Promega) 1 μl
10x Buffer D (Promega) 3 μl
25.5 μl of H ₂ O

  The plasmid was digested for about 1 hour at 37 ° C. The linear plasmid fragment was purified by agarose gel electrophoresis, recovered from the gel using Qiagen's QIAquick gel extraction kit (spin column), and recovered in 50 μl of elution buffer. 5 μl BamHI buffer (New England Biolabs) and 10 units BamHI enzyme were added. The plasmid was digested for about 1 hour at 37 ° C. The desired vector fragment was purified by agarose gel electrophoresis, recovered from the gel using Qiagen's QIAquick gel extraction kit (spin column), and recovered in 50 μl of elution buffer.

Ligation of NtermEGFP-NZ fragment to pEGFP-C1 and ligation of CZ-CtermEGFP fragment to PS0609
1 μl of cut and purified vector fragment
Cleaved and purified NtermEGFP-NZ or CtermEGFP fragment
3 μl
10xT4 DNA ligase buffer (New England Biolabs)
1 μl
T4 DNA ligase (400 U / μl, New England Biolabs)
0.5 μl
5 μl of H ₂ O

  Incubate the ligation reaction overnight at 16 ° C. 3 μl was transformed into One Shot TOP10 chemically competent E. coli cells (Invitrogen) and transformants were selected for pEGFP-C1 and PS0609 derivatives on kanamycin-added immedia or zeocin-added immedia (both Invitrogen), respectively. .

  Four transformants (from each transformation plate) were grown for 6 hours at 37 ° C. in immedia media plus appropriate selection (kanamycin or zeocin).

  Plasmid DNA was isolated by QIAprep spin column method (Qiagen) and analyzed by restriction enzyme digestion with AseI and MluI. The DNA sequence of the insert was finally verified by sequencing (as described above). The resulting plasmids were named PS1557, PS1558, PS1559, and PS1560 (Table 4).

Example 7: EGFP-based reconstitution in mammalian cells
To establish cell lines expressing EGFP fragment / zipper fusion proteins, CHO-hIR cells were transformed with plasmid pairs and two cell lines 1) CHO-hIR PS1559 + PS1557, and 2) CHO-hIR. PS1560 + PS1558 was created. The CHO-hIR cell line consists of CHO-K1 (ATCC CCL-61) cells, which are stably transfected with human insulin receptor ((hIR, GenBank Acc #; M10051) as described in Hansen, BF, Danielsen, GM, Dreamer, K., Srensen, A.R., Wiberg, F.C., Klein, H.H., Lundemose, A.G. 1996) Sustained signaling from the insulin receptor after stimulation with insulin analogues exhibiting incimated mitogen potency. pr 1; 315 (Pt 1): 271-279) <. The selectable marker for the vector is methotrexate (MTX). hIR expression is very stable in CHO-hIR cells (without sputum pressure) because the cell line is insulin sensitive and can maintain a very stable phenotype without sputum pressure.

  Stable cells were obtained by growing cells in selective medium (containing geneticin and zeocin).

  CHO-hIR was transfected with Fugene (Roche) (according to manufacturer's instructions). The day after transfection, the cells were examined for transient expression, split (1:10), selective medium (500 μg / ml Geneticin (Invitrogen) and 1 mg / ml Zeocin (Cayla)). To the growth medium). The cell line was stable after 2-3 weeks of culture in selective medium.

  The growth medium used was NUT. MIX F-12 (Ham's) withGLUTAMAX-1 (Gibco / Invitrogen) was supplemented with 10% fetal calf serum (JRH Biosciences) and 1% penicillin-streptomycin (10,000 IU / ml, Gibco / Invitrogen) Medium. The CHO-hIR cells were cultured in growth medium and split (1: 4 to 1:16) (twice a week according to standard cell culture protocol). The CHO-hIR PS1559 + PS1557 and CHO-hIR PS1560 + PS1558 were treated similarly (except that the growth medium was always supplemented with 500 μg / ml Geneticin (Invitrogen) and 1 mg / ml Zeocin (Cayla)).

  3 CHO-hIR cell lines, which include pEGFP-C1 (expressing EGFP with one short C-terminal extension), PS1559 + PS1557 (EGFP complementary fragment divided by 157-158, NtermEGFP157-NZ + CZ-CtermEGFP158 expressing) and PS1560 + PS1558 (EGFP complementary fragment divided by 172-173, NtermEGFP172-NZ + CZ-CtermEGFP173 expressing separately) images after transfection 1 The relative brightness of cells that were collected at day 2, day 2 and day 10 and expressing different complementation constructs were evaluated. Images were collected on a Nikon Diaphot 300 (equipped for epifluorescence work). The light source for epifluorescence used a Nikon 100W Hg arc lamp, which was connected to a microscope through a custom quartz fiber illuminator (TILL Photonics GmbH, Planegg, Germany). Excitation light passes through a 450-490 nm bandpass filter (Delta Light and Optics, Lyngby, Denmark) and the specimen is Chroma 72100 505 nm cut-on dichroic mirror (Chroma Technology, BrattleVorT, US). ) Directed through. An Ax40 NA1.3 oil immersion lens was used for all images. The emitted light reached the Hammamatsu Orca ER camera through a 540-550 bandpass filter (Chroma). All images are collected with a 50 ms exposure time and image desaturation is assured even for cells of maximum brightness (EGFP expression) in each optical field (maximum pixel count <4095). Selected to be. The imaging software used to acquire images in this system was IPLab for Windows (Scananalytics, USA).

Image presentation and analysis
Microscopic images were analyzed using the ImageJ software package, which is public domain image analysis software, created by Wayne Rasb of the National Institutes of Health (http://rsb.info.nih.gov/ij/). And data analysis was performed with Microsoft Excel.

  The image shown in FIG. 2 is an image of fluorescent CHO-hIR cells co-transfected with different NtermEGFP-NZ and CZ-CtermEGFP expression vectors or transfected with pEGFP-C1. The images are individually scaled and the cells and the fluorescence distribution therein are visualized. Because of this scaling, the relative fluorescence levels cannot be compared between images. When the same images are measured idly, they appear as in FIG. It is also clear that cells transfected with a complementary construct based on the split between residues 172 and 173 are significantly more fluorescent than cells transfected with a complementary construct based on the split between residues 157 and 158 It is. However, cells transfected with the pEGFP-C1 construct exhibit significantly stronger fluorescence on day 2.

  The same image was analyzed for background and maximum fluorescence intensity using the ImageJ software package (Figure 4). From the figure it is clear that the split between residues 172 and 173 (and possibly anotherwhere in this loop) is the split between residues 157 and 158 (and maybe This is very superior to (division in another area in the loop).

Example 8: Eukaryotic Expression Vector Encoding EYFP and EYFP Variant F64L Fragment / Zipper Fusion Protein
Mutagenesis of the eukaryotic NtermEGFP-NZ expression vectors PS1559 (NtermEGFP157-NZ) and PS1560 (NtermEGFP172-NZ) to the corresponding N-terminal EYFP (SEQ ID NO: 5) fragment (NtermEYFP-NZ) variants, and Mutagenesis of the eukaryotic CtermEGFP expression vectors PS1557 (CZ-CtermEGFP158) and PS1558 (CZ-CtermEGFP173) to the corresponding C-terminal EYFP fragment (CZ-CtermEYFP) variant is site-specific abrupt using the QuickChange kit This was achieved by mutagenesis according to the manufacturer's instructions (Stratagene). Primers 2333 and 2334 were used to convert expression vectors PS1559 (NtermEGFP157-NZ) and PS1560 (NtermEGFP172-NZ) into N-terminal EYFP fragment expression vectors PS1639 (NtermEYFP157-NZ) and PS1642 (NtermEYFP172-NZ). . The introduced mutation was: L64F: T65G: V68L: S72A. Furthermore, using primers 2335 and 2336, the expression vectors PS1559 (NtermEGFP157-NZ) and PS1560 (NtermEGFP172-NZ) were F64L-mutated N-terminal EYFP fragment expression vectors PS1640 (NtermE [F64L] YFP157-NZ) and PS1641 (NtermE [F64L] YFP172-NZ). The introduced mutation was: T65G: V68L: S72A. Thus, the expressed NtermEYFP fragment has the following amino acid sequence (only residues 64-72 are shown):

  Finally, using primers 2337 and 2338, expression vectors PS1557 (CZ-CtermEGFP158) and PS1558 (CZ-CtermEGFP173) were transferred to C-terminal EYFP fragment expression vectors PS1637 (CZ-CtermEYFP158) and PS1638 (CZ-CtermEYFP173), It was converted by introducing a mutation. The entire sequence was verified by DNA sequencing of the vector. All primer sequences are shown in Table 2.

Example 9: EYFP-based reconstitution in mammalian cells
The constructed EYFP-based split fluorescent protein expression vectors PS1637 to PS1642 (above) were investigated in mammalian cells in parallel with the EGFP-based split fluorescent protein expression vectors PS1557 to PS1560 (described in Example 7), where The same experimental setup (including the same filter set) and processing (including image analysis processing) were used, but all images were generated using a 10 ms exposure time (instead of a 50 ms exposure time). This is because the brightness increased and a 20x objective was used instead of a 40x objective to image more cells. Other suitable filter sets could be used. Images are acquired the day after transfection (Day 1).

  Similarly, from the fluorescence image of the transfected cells measured (FIG. 6), it is again shown that the split site between residues 172 and 173 is superior to the split site between residues 157 and 158. It is clear that it is shown. Moreover, it is clear that complementarity based on EYFP fragments is superior to complementarity based on EGFP fragments. Surprisingly, the introduction of the F64L mutation from EGFP into the N-terminal EYFP fragment further enhanced the fluorescence of the complementary fragment. As can be seen from the image, using the optimal split site (between residues 172 and 173), using the optimal fluorescent protein color variant (EYFP) and introducing the F64L folding mutation into the NtermEYFP fragment The positive effect of, is additive. Quantification of these observations was performed by analyzing the image shown in FIG. 6 (the numerical output is represented in FIG. 7).

  It is interesting that the optimal constructs (NtermE [F64L] YFP172-NZ and CZ-CtermE [F64L] YFP173) are as strong as EYFP when reconstituted. A large increase in fluorescence intensity increases the amount of noise to signal in many types of quantitative cell analysis (eg, high-throughput screening and microscopy), so low amounts of Important in order to facilitate the detection of probes in vivo or in vitro.

  Also, functional fluorescent complexes (of potentially different colors) can be produced by mixing NtermEYFP with CtermEGFP or NtermEGFP with CtermEYFP fragments (FIGS. 8 and 9).

  Fragments with overlapping sequences are equally functional and are, for example, functional cloning systems where the highly flexible linker sequence is due to the very diverse nature of the fusion partner It is very attractive in the required system. The overlapping fragments allow any of the fusion partners to have a long linker sequence (quantified in FIGS. 8 and 9).

Example 10: Construction of PS1769, 1767, 1771, 1768
The plasmid PS1769 is a fusion in which NtermE [F64L] YFP172 and FKBP are linker sequences GSGSGSGTITSLYKKAGST (single letter amino acid code, SEQ ID NO: 11), partially linked by a linker sequence derived from Gateway recombination sequences. Code the body.

  In the plasmid PS1767, NtermE [F64L] YFP172 and the FKB FKBP binding part [FRB (amino acids 2025-2114 of FRAP)] are the linker sequence GSSGSGDITSLYKKAGST (single-letter amino acid code, SEQ ID NO: 12). It encodes a fusion linked by a linker sequence derived from a Gateway recombination sequence.

  Plasmid PS1771 encodes a fusion in which FRB and CtermEYFP173 are linker sequences DPAFLYKVVISGSGSSG (one-letter amino acid code, SEQ ID NO: 13), partially linked by a linker sequence derived from Gateway recombination sequences To do.

  Plasmid PS1768 encodes a fusion in which FKBP and CtermEYFP173 are linker sequences DPAFLYKVVISSGGSSG (single letter amino acid code, SEQ ID NO: 14), partially linked by a linker sequence derived from Gateway recombination sequences. To do.

  Construction of plasmid PS1769

  Plasmid PS1769 has NtermE [F64L] YFP172 and FKBP linked by a linker sequence, placed under the control of a CMV promoter, and has kanamycin and neomycin resistance as selectable markers in E. coli and mammalian cells, respectively. , Coding the fusion.

  Plasmid PS1769 was derived from plasmids PS1799 (entry clone) and PS1679 (destination vector). Plasmid PS1679 was derived from plasmid PS1672 and pEGFP-C1 (Clontech). Plasmid PS1672 was derived from plasmid PS1641 described above.

  Construction of intermediate PS1672

  PS1641 was subjected to PCR with primers 2219 and 2222 (Table 2) and the ca 0.5 kb Nhe1-BamH1 fragment was ligated into pEGFP-C1 (Clontech) digested with Nhe1 and BamH1. This replaces NtermEGFP with NtermE [F64L] YFP172, followed by a linker sequence (which encodes the in-frame linker sequence Gly-Ser-Gly-Ser-Gly-Ser-Gly). And a unique EcoRV site (just upstream of BamH1) is placed. This plasmid is called PS1672.

  Construction of destination vector PS1679

  Plasmid PS1672 was transformed into a Gateway compatible destination vector by cutting the DNA with EcoRV and ligating it with Gateway cassette reading frame A [according to Gateway manufacturer (Invitrogen) instructions]. . This destination vector is called PS1679.

  Construction of Gateway entry clone PS1779

  The coding sequence for FKBP (GenBank Acc no XM — 016660) was isolated from human cDNA using PCR and primers 2442 and 1272 (Table 2). The ca 0.4 kb product was transferred to the donor vector pDONR207 by BP reaction (according to the manufacturer's recommendations (Invitrogen)) to produce the entry clone PS1779.

  Finally, the expression vector PS1769 was produced by transferring FKBP from the entry clone PS1779 to the destination vector PS1679 by LR reaction (according to the manufacturer's recommendations (Invitrogen)).

  Construction of plasmid PS1767

  Plasmid PS1767 is composed of NtermE [F64L] YFP172 and FRAP's FKBP binding moiety [FRB (amino acids 2025-2114 of FRAP)] linked by a linker sequence, placed under the control of the CMV promoter, and resistant to kanamycin and neomycin. It encodes fusions that each have as a selectable marker in E. coli and mammalian cells.

  Plasmid PS1767 was derived from plasmids PS1781 (entry clone) and PS1679 (destination vector). Plasmid PS1679 was constructed as described above.

  Construction of Gateway entry clone PS1781

  The FKBP binding portion of FRAP (amino acids 2025-2114, Gen Bank Acc no XM — 001528) was separated from human cDNA using PCR and primers 2444 and 1268 (Table 2). The ca 0.3 kb product was transferred to the donor vector pDONR207 by BP reaction (according to the manufacturer's recommendations (Invitrogen)), producing the entry clone PS1781.

  Finally, expression vector PS1767 was produced by transferring FRB from entry clone PS1781 to destination vector PS1679 by LR reaction (according to manufacturer's recommendations (Invitrogen)).

  Construction of plasmid PS1771

  In the plasmid PS1771, the FKB FKBP binding part (FRA amino acids 2025-2114) called FRB and the EYFP C-terminal (FRB-CtermEYFP173) are linked by a linker sequence and placed under the control of the CMV promoter. It encodes a fusion that has zeocin resistance as a selectable marker in E. coli and mammalian cells.

  Plasmid PS1771 was derived from plasmids PS1782 (entry clone) and PS1688 (destination vector). Plasmid PS1688 was derived from plasmids PS1674 and PS609 described above. Plasmid PS1674 was derived from plasmid PS1638 described above.

  Construction of intermediate PS1674

  PS1638 was subjected to PCR with primers 2225 and 2132 (Table 2) and the ca 0.25 kb Nhe1-BamH1 fragment was ligated to PS609 digested with Nhe1 and BamH1. This replaces EGFP with EYFP (173-238), preceded by a linker sequence (which encodes the in-frame linker sequence Gly-Ser-Gly-Ser-Gly-Ser-Gly). And a unique EcoRV site (just downstream of Nhe1). This plasmid is called PS1674.

  Construction of destination vector PS1688

  Plasmid PS1674 was converted to a Gateway compatible destination vector by cutting the DNA with EcoRV and ligating it with Gateway cassette reading frame A [according to Gateway manufacturer (Invitrogen) instructions]. . This destination vector is called PS1688.

  Construction of Gateway entry clone PS1782

  The FKBP binding portion of FRAP (Gen Bank Acc no XM — 001528, amino acids 2025-2114) was isolated from human cDNA using PCR and primers 2444 and 2445 (Table 2). The ca 0.3 kb product was transferred to the donor vector pDONR207 by BP reaction (according to the manufacturer's recommendations (Invitrogen)), producing the entry clone PS1782.

  Finally, expression vector PS1768 was produced by transferring FRB from entry clone PS1782 to destination vector PS1688 by LR reaction (according to manufacturer's recommendations (Invitrogen)).

  Construction of plasmid PS1768

  Plasmid PS1768 is a fusion in which FKBP and EYFP (173-238) (FKBP-CtermEYFP173) are placed under the control of the CMV promoter and have zeocin resistance as a selectable marker in E. coli and mammalian cells. Is encoded.

  Plasmid PS1768 was derived from plasmids PS1780 (entry clone) and PS1688 (destination vector). Plasmid PS1688 was constructed as described above.

  Construction of Gateway entry clone PS1780

  The FKBP coding sequence (GenBank Acc no XM — 016660) was isolated from human cDNA using PCR and primers 2442 and 2443 (Table 2). The ca 0.4 kb product was transferred to the donor vector pDONR207 by BP reaction (according to the manufacturer's recommendations (Invitrogen)), producing the entry clone PS1780.

  Finally, the expression vector PS1768 was produced by transferring FKBP from the entry clone PS1780 to the destination vector PS1688 via the LR reaction (according to the manufacturer's recommendations (Invitrogen)).

  Example 11: Construction of an inducible interaction system using a GFP complementation method (demonstrating the utility of the method in screening for compounds that inhibit protein-protein interactions).

  Immunosuppressive compound Rapamycin binds to FK506 binding protein (FKBP) and simultaneously binds to the large Pl3 kinase homologue FRAP (also known as mTOR or RAFT) as a heterodimerizer for these two proteins Works. To induce heterodimers between desired proteins using rapamycin, one of the proteins is in the FKBP domain and the other is the 90 amino acid portion of FRAP (referred to as FRB, FKBP-rapamycin complex). (Chen et al, PNAS 92, 4947 (1995)). In this example, the fusion of FRB and FKBP is complementary to the complementary half of the split EYFP, which includes the F64L mutation in the EYFP (1-172) sequence (NtermE [F64L] YFP172). A sexual response is created that can be controlled by the addition of rapamycin.

  This example demonstrates that a model GFP complementation system using components that can be created to interact conditionally responds to interactive stimuli in a dose-dependent manner as expected. The examples also provide information about the rate of fluorescence development for the E [F64L] YFP complementation system. It is further demonstrated that the system can be used to detect compounds that block protein interactions fused to the complementary half of the E [F64L] YFP complementation system.

  The following fusion constructs were generated as described in Example 10:

NtermE [F64L] YFP172-FKBP = plasmid code PS1769
FRB-CtermEYFP173 = PS1771
NtermE [F64L] YFP172-FRB = PS1767
FKBP-CtermEYFP173 = PS1768
Probes were co-transformed into CHO-hIR cells (supra) in pairs, with PS1771 as PS1771 and PS1767 as PS1768, transfection factor FuGENE ^™ Corp. (Boehringer Mannheim Corp, USA). Was used in accordance with the method recommended by suppliers. Cells in growth medium (with HAM's F12 nutrient mixture, Glutamax-1, 10% fetal bovine serum (FBS), 100 μg penicillin-streptomycin mixture ml ⁻¹ (supplied by GibcoBRL, Life Technologies, Denmark) In culture. Transfected cells were cultured in this medium with the addition of two selection agents appropriate for the plasmid used, 1 mg / ml zeocin plus 0.5 mg / ml G418 sulfate. Cells were cultured at 37 ° C. with 100% humidity and normal atmosphere supplemented with 5% CO ₂ .

  After culturing for 10-12 days in the continuous presence of the selective agent, it was determined whether the resulting cell line was stably transfected. For fluorescence microscopy, aliquots of cells are transferred to Lab-Tek chambered cover glasses (Nalge Nunc International, Naperville USA) and allowed to attach for at least about 24 hours until they reach about 80% confluence. Oita. Images are collected as specified using a Nikon Diaphot 300 inverted fluorescent microscope (Nikon Corp., Tokyo, Japan), x20 (dry) and / or x40 (oil-immersed) objectives, and Orca ER charged coupler (CCD) camera (Hammamatsu Photonics KK, Hammamatsu City, Japan). Cells were illuminated with a 100 W HBO arc lamp, via a 470 ± 20 nm excitation filter, a 510 nm dichroic mirror and a 515 ± 15 nm emission filter (for minimal image background). Image collection, subsequent fluorescence intensity measurement and analysis were all controlled by specific software (IPLab Spectrum for Windows software, Scanatics, Fairfax, VA USA).

Also, cells are cultured for 16 hours and have approximately 1.0 × 10 ⁵ cells / 400 μL in a plastic 96-well plate (Polyfiltronics Packard 96-View Plate or Costar Black Plate, clear bottom; both types have been tissue culture treated) From the seeding density, it was used for both imaging purposes and measurement of fluorescence intensity in a fluorescence plate reader. Prior to the experiment, the cells are cultured overnight in Ham's F-12 medium (with glutamax, 100 μg penicillin-streptomycin mixture ml-1 and 10% FBS) without selective agents. In cells, the medium has low autofluorescence, which allows for the measurement of straight from the incubator fluorescence. For endpoint measurements, cells in the plate were fixed routinely (4% formaldehyde in phosphate buffered saline (PBS) + 10 μM Hoechst 22538 for 10 minutes), then 3 times with PBS. A washing step was performed. The use of the nuclear dye Hoechst 22538 allows the EYFP fluorescence signal from each well to be corrected for cell density. Plates prepared in this way are run on a device equipped with a suitable filter set (EYFP: excitation 485 nm, emission 527 nm; Hoechst 22538: excitation 355 nm, emission 460 nm) in a Fluoroskan Ascent CF plate reader (Labsystems, Finland). It was measured.

  Both cell lines CHO-hIR [PS1769 + PS1771] and CHO-hIR [PS1767 + PS1768] responded to rapamycin and showed a substantial increase in EYFP fluorescence as expected after several hours of incubation (FIG. 10). At the starting conditions for these cells (t = 0), fluorescence is barely visible in most cells, but nevertheless some cells (<5%) in the population are low (but It was noted that it had appreciable fluorescence before treatment (FIG. 10a). After 4 hours (FIG. 10b), many cells (approximately 40%) developed significantly higher EYFP fluorescence throughout the cytoplasmic and nuclear compartments. After 16 hours (FIG. 10c), the cell-by-cell response increased further and encompassed a large proportion (about 70%) of the cell population. The results were essentially the same for the second cell line CHO-hIR [PS1769 + PS1771].

  The graph of FIG. 11 shows the rate of generation of EYFP fluorescence in cells after the rapamycin treatment of the CHO-hIR [PS1767 + PS1768] strain. Cells were treated with 3 μM rapamycin in 96 well plates and fluorescence was measured at various times. Treatments and measurements were performed on cells grown in Ham's medium + 10% FBS and fluorescence measurements were corrected for background fluorescence from this medium. The graph demonstrates that the half-time for fluorescence generation is about 5 hours. The rate of fluorescence generation includes time for the interaction between FKBP and FRB mediated by the dimerizer rapamycin, plus time for annealing of the EYFP moiety (moieties), and successfully annealed EYFP protein Included is the (possibly very long) time required for the maturation of the inner fluorophore.

  FIG. 12 is a response curve for CHO-hIR [PS1769 + PS1771] cell lines at different rapamycin doses. Cells were cultured in 96-well plates, treated with various rapamycin doses for 16 hours, then fixed and stained with Hoechst before EYFP fluorescence / cells (arbitrary units) were determined on an Ascent plate reader. Values are corrected for PBS background and cell number. The cell line shows an approximately 3-fold increase in EYFP intensity / cell over the rapamycin dose range used in this experiment.

  One mode of increasing the dynamic range of the response and reducing the inherent EYFP background signal from these cell lines is to allow the fraction of cells that emit the EYFP bright to undergo a rapamycin stimulation prior to rapamycin stimulation. Is to remove. This is easily accomplished by fluorescence activated cell sorting (FACS) method. Each cell line was sorted into three groups by this method: (i) the most green group (ii) moderate to low green group and (iii) black group. The “most green” was discarded in each case, while the other two groups were cultured for further use. FIGS. 13 (a) and 13 (b) show the improved response of the cell line CHO-hIR [PS1767 + PS1768] to 100 nM rapamycin after the sorting procedure.

  FIGS. 14 (a) and (b) show the responses of the “moderate to low green” and “black” FACS groups (respectively) derived from the CHO-hIR [PS1767 + PS1768] parent strain. The dose response to rapamycin was measured after 7 hours (a) and 30 hours (b) for each cell line.

  Fluorescence values were corrected for plate & media background. The increase in EYFP fluorescence is over 20 times the unstimulated value in each case.

Unexpectedly, the cells are still alive during this period, but the absolute fluorescence signal does not appear to change significantly between 7 and 30 hours. Furthermore, the dose response curves at 7 and 30 hours for each cell line are very close and similar, with about 0.25 μM in the “moderate to low green” group and 0.1 μM in the “ The EC ₅₀ in the “black” group. This data suggests that once dimerization occurs, EYFP complementation is stable in the cell for over 30 hours. The “moderate to low green” group has a larger overall response range, reaching 3 times higher intensity than the black group at the maximum rapamycin concentration. Both FACS groups have significantly lower pre-stimulated fluorescence intensity compared to the parent (non-FACS'd) strain

FIGS. 15 (a) and (b) show two FACS′d strains, CHO-hIR [PS1768 + PS1767] “moderate to low green” groups (FIG. 15 (a)) and CHO− for FK506 versus 100 nM rapamycin. The dose response competition curve in hIR [PS1769 + PS1771] "black" group (FIG.15 (b)) is shown. The EC ₅₀ value for both cases is approximately 1.2 μM FK506. Cells were incubated overnight (16 hours) with a mixture of the two compounds, then fixed and stained with Hoechst on an Ascent plate reader prior to EYFP fluorescence / cell determination. Plate and solution backgrounds are subtracted; the dotted line on each graph indicates the level of fluorescence before stimulation for each cell line in these experiments. These can be successfully screened for compounds that interfere with the conditional interaction between the two protein components using the GFP complementation method using fusions to NtermE [F64L] YFP172 and CtermEYFP173. The results point out.

Example 12: Construction of PS1736, PS1758, PS1706, PS1810, PS1809, and PS1827
Plasmid PS1736 is a fusion in which F64L, NY172-NZ, and H2B are linker sequences GSGSGSGSITTSLYKKAGST (single letter amino acid code, SEQ ID NO: 74), partially linked by a linker sequence derived from a Gateway recombination sequence. Is encoded.

  Plasmid PS1758 is a fusion in which beta-catenin and CZ-CY173 are linked by a linker sequence DPAFLYKVVISSGSGSG (amino acid code of one letter code, SEQ ID NO: 69) partially derived from Gateway recombination sequences Is encoded.

Plasmid PS1706 is a linker sequence in which F64L, NY172 (= NtermE [F64L] YFP172) and beta-catenin are linker sequences GSGSGSGSITTSLYKKAGST (amino acid code of one letter code, SEQ ID NO: 70), partially derived from Gateway recombination sequences Encodes a fusion linked by
Plasmid PS1810 encodes a fusion of H2B and TCF4 (1-70) and CY173 (CtermEYFP173), where H2B and TCF4 (1-70) are the linker sequence DITSLYKKAGST (SEQ ID NO: 75) Linked by a linker sequence derived from the Gateway recombination sequence, where TCF4 (1-70) and CY173 (CtermEYFP173) are the linker sequences DPFLYKVVISSGSGSG (single letter amino acid code, SEQ ID NO: 71), which are partially Gateway Linked by a linker sequence derived from the recombinant sequence.

  Plasmid PS1809 encodes F64L, NY172 (NtermE [F64L] YFP172) and a fusion of FKBP and betacatenin, where F64L, NY172 (NtermE [F64L] YFP172) and FKBP are the linker sequences GSGSGSGDDL (one-letter amino acids) Linked by the code, SEQ ID NO: 72), where FKBP and beta-catenin are the linker sequence GTGTGTGDITSLYKKAGST (SEQ ID NO: 76), linked in part by a linker sequence derived from the Gateway recombination sequence.

  Plasmid PS1827 encodes a fusion of the FKBP binding portion of H2B and FRAP (amino acids 2025-2114) and CY173 (CtermEYFP173), where the FKBP binding portion of H2B and FRAP (amino acids 2025-2114) is the linker sequence DPAFLYKVVISGTGTTGTG (1 The amino acid code in letter code, SEQ ID NO: 73), partially linked by a linker sequence derived from the Gateway recombination sequence, wherein the FKB FKBP binding portion (amino acids 2025-2114) and CY173 (CtermEYFP173) of FRAP are linkers It is linked by the array LPSGSGSGSG (SEQ ID NO: 77).

  Construction of plasmid PS1736

  Plasmid PS1736 has F64L, NY172-NZ and H2B linked by a linker sequence and placed under the control of a CMV promoter and has kanamycin and neomycin resistance as selectable markers in E. coli and mammalian cells, respectively. Encodes the fusion.

  Plasmid PS1736 was derived from plasmids PS1667 (entry clone) and PS1695 (destination vector). Plasmid PS1695 was derived from plasmid PS1673. Plasmid PS1673 was derived from plasmid PS1641 and pEGFP-C1 (Clontech) described above. Plasmid PS1672 was derived from plasmid PS1641 described above.

  Construction of intermediate PS1673

  PS1641 was subjected to PCR with primers 2219 and 2387 (Table 2) and the ca 0.5 kb Nhe1-BamH1 fragment was ligated into pEGFP-C1 (Clontech) digested with Nhe1 and BamH1. This replaces EGFP with F64L, EYFP (1-172), followed by a linker sequence (which encodes the in-frame linker sequence GGTGSG-NZ-GSSGSGSG) and a unique EcoRV site. (Just upstream of BamH1) is arranged. This plasmid is called PS1673.

  Construction of destination vector PS1695

  Plasmid PS1673 was converted to a Gateway compatible destination vector by cutting the DNA with EcoRV and ligating it with Gateway cassette reading frame A [according to Gateway manufacturer (Invitrogen) instructions]. . This destination vector is called PS1695.

  Construction of Gateway entry clone PS1667

  The coding sequence for H2B (GenBank Acc no NM_003518) was isolated from human cDNA using PCR and primers 2367 and 2368 (Table 2). The ca 0.4 kb product was transferred to the donor vector pDONR207 by BP reaction (according to the manufacturer's recommendations (Invitrogen)) to produce entry clone PS1667.

  Finally, expression vector PS1736 was produced by transferring H2B from entry clone PS1667 to destination vector PS1695 by LR reaction (according to manufacturer).

  Construction of plasmid PS1758

  Plasmid PS1758 is a fusion in which beta-catenin and CZ-CY173 are linked by a linker sequence and placed under the control of a CMV promoter and have zeocin resistance as a selectable marker in E. coli and mammalian cells. It is coded.

  Plasmid PS1771 was derived from plasmids PS1762 (entry clone) and PS1696 (destination vector). Plasmid PS1696 was derived from plasmid PS1726. Plasmid PS1726 was derived from plasmids PS1638 and PS609 described above.

  Construction of intermediate PS1726

  PS1638 was subjected to PCR with primers 2388 and 2132 (Table 2) and the ca 0.35 kb Nhe1-BamH1 fragment was ligated to PS609 digested with Nhe1 and BamH1. This replaced EGFP with EYFP (173-238), preceded by a linker sequence (which encodes the in-frame linker sequence GSGSSGSG-zip) and a unique EcoRV site (Nhe1). Just downstream) is arranged. This plasmid is called PS1726.

  Construction of destination vector PS1696

  Plasmid PS1726 was converted to a Gateway compatible destination vector by cutting the DNA with EcoRV and ligating it with Gateway cassette reading frame A [according to Gateway manufacturer instructions (Invitrogen) instructions]. . This destination vector is called PS1696.

  Construction of Gateway entry clone PS1762

  The coding sequence for beta-catenin (GenBank Acc no X87838) was isolated from human cDNA using PCR and primers 2359 and 2439 (Table 2). The ca 2.3 kb product was transferred to the donor vector pDONR207 by BP reaction (according to the manufacturer's recommendations (Invitrogen)), producing the entry clone PS1762.

  Finally, the expression vector PS1758 was produced by transferring the beta catenin coding sequence from the entry clone PS1762 to the destination vector PS1696 by LR reaction (according to the manufacturer's recommendations (Invitrogen)).

  Construction of plasmid PS1706

  Plasmid PS1706 is a fusion in which F64L, NY172 and beta-catenin are linked by a linker sequence and placed under the control of a CMV promoter and have kanamycin and neomycin resistance as selectable markers in E. coli and mammalian cells, respectively. The body is coded.

  Plasmid PS1706 was derived from plasmids PS1664 (entry clone) and PS1679 (destination vector). Plasmid PS1679 is described above.

  Construction of Gateway entry clone PS1664

  The coding sequence of beta catenin (GenBank Acc no X87838) was isolated from human cDNA using PCR and primers 2359 and 2360 (Table 2). The ca 2.3 kb product was transferred to the donor vector pDONR207 by BP reaction (according to the manufacturer's recommendations (Invitrogen)), producing the entry clone PS1664.

  Finally, the expression vector PS1706 was produced by transferring beta-catenin from the entry clone PS1664 to the destination vector PS1679 by LR reaction (according to the manufacturer's recommendations (Invitrogen)).

  Construction of plasmid PS1810

  Plasmid PS1810 has H2B-TCF4 (1-70) and CY173 linked by a linker sequence and placed under the control of a CMV promoter, and has zeocin resistance as a selectable marker in E. coli and mammalian cells. , Coding the fusion.

  Plasmid PS1810 was derived from plasmids PS1669 (entry clone) and PS1806 (destination vector). Plasmid PS1806 was derived from plasmid PS1790. Plasmid PS1790 was derived from plasmid PS1674 described above.

  Construction of intermediate PS1790

  The coding sequence for H2B (GenBank Acc no NM — 003518) was isolated from human cDNA using primers 2389 and 2390 (Table 2) and ligated to PS1674 digested with ca 0.4 kb Nhe1-EcoRV with Nhe1 and EcoRV. This replaced the H2B upstream of EYFP (173-238), preceded by a linker sequence (which encodes the in-frame linker sequence GSGSSGSG-zip), and H2B and linker EYFP ( 173-238) between the unique EcoRV sites. This plasmid is designated PS1790.

  Construction of destination vector PS1806

  Plasmid PS1790 was converted into a Gateway compatible destination vector by cutting the DNA with EcoRV and ligating it with Gateway cassette reading frame A [according to the instructions of the Gateway manufacturer (Invitrogen)]. . This destination vector is called PS1806.

  Construction of Gateway entry clone PS1669

  The coding sequence for the N-terminal 70 amino acids of TCF4 (GenBank Acc no Y11306) was isolated from human cDNA using PCR and primers 2364 and 2366 (described below). The ca 0.25 kb product was transferred to the donor vector pDONR207 via a BP reaction (according to the manufacturer's recommendations (Invitrogen)), producing the entry clone PS1669.

  Finally, the expression vector PS1810 was produced by transferring the coding sequence of TCF4 (1-70) from the entry clone PS1669 to the destination vector PS1806 by LR reaction (according to the manufacturer's recommendations (Invitrogen)) .

  Construction of plasmid PS1809

  Plasmid PS1809 has F64L, NY172 and FKBP-betacatenin linked by a linker sequence and placed under the control of a CMV promoter and has kanamycin and neomycin resistance as selectable markers in E. coli and mammalian cells, respectively. , Coding the fusion.

  Plasmid PS1809 was derived from plasmids PS1664 (entry clone) and PS1805 (destination vector) described above. Plasmid PS1805 was derived from plasmid PS1789. Plasmid PS1789 was derived from plasmid PS1672 above and PS1768 above.

  Construction of intermediate PS1789

  PS1768 was subjected to PCR with primers 2462 and 2463 and the ca 0.35 kb fragment was digested with Pvu2 and BamH1 and ligated to PS1672 digested with EcoRV and BamH1. This produces a fusion in which F64L, NY172 and FKBP are joined by a GSGSGSGDDL linker, which has a GTGTTGTG linker sequence (behind FKBP) and a unique EcoRV site (just downstream of the GT3 linker). This plasmid is called PS1789.

  Construction of destination vector PS1805

  Plasmid PS1789 was converted into a Gateway compatible destination vector by cutting the DNA with EcoRV and ligating it with Gateway cassette reading frame A [according to Gateway manufacturer (Invitrogen) instructions]. . This destination vector is called PS1805.

  Finally, the expression vector PS1809 was produced by transferring beta-catenin from the entry clone PS1664 to the destination vector PS1805 by LR reaction (according to the manufacturer's recommendations (Invitrogen)).

Construction of plasmid PS1827
Plasmid PS1827 has H2B and FRAP FKBP binding moieties (amino acids 2025-2114) and CY173 linked by a linker sequence and placed under the control of the CMV promoter, making zeocin resistance a selectable marker in E. coli and mammalian cells. Has a fusion encoding.

  Plasmid PS1827 was derived from plasmids PS1732 (entry clone) and PS1826 (destination vector). Plasmid PS1826 was derived from plasmid PS1788. Plasmid PS1788 was derived from plasmids PS1674 and PS1767 described above.

  Construction of intermediate PS1788

  PS1767 was subjected to PCR with primers 2464 and 2465 and the ca 0.35 kb product was digested with Nhe1-Sma1 and ligated to PS1674 digested with Nhe1 and EcoRV.

  This produced a fusion in which FRAP (2025-2114) and EYFP (173-238) were linked by a linker sequence (GSGSGSSG), which had a GGTTGTG linker sequence (before FRAP) and a unique EcoRV site (GTGTTGTG). Has just upstream of the linker). This plasmid is called PS1788.

  Construction of destination vector PS1826

  Plasmid PS1788 was converted into a Gateway compatible destination vector by cutting the DNA with EcoRV and ligating it with Gateway cassette reading frame A [according to Gateway manufacturer instructions (Invitrogen) instructions]. . This destination vector is called PS1826.

  Construction of Gateway entry clone PS1732

  The coding sequence of H2B (GenBank Acc no NM_003518) was isolated from human cDNA using PCR and primers 2367 and 2407 (Table 2). The ca 0.4 kb product was transferred to the donor vector pDONR207 by BP reaction (according to the manufacturer's recommendations (Invitrogen)) to produce the entry clone PS1732.

  Finally, expression vector PS1827 was produced by transferring H2B from entry clone PS1732 to destination vector PS1826 by LR reaction (according to manufacturer's recommendations (Invitrogen)).

  Example 13: Assay for screening in living cells for compounds that modify the translocation behavior of one or both members of a target protein pair and / or the interaction between those proteins.

  This example demonstrates the construction of a protein translocation assay using the GFP complementation method, which modifies the translocation behavior of one or both members of a target protein pair and / or the interaction between those proteins The usefulness of the method in screening in vivo for compounds that do (translocation and interaction dependent complementation, TIDC) is demonstrated.

  This example demonstrates that: a GFP complementation system can be designed to report the degree of interaction between one protein and a partner protein in a living cell, specifically Can only cause such interactions to occur after translocation of one or both proteins (which brings both components in close proximity to one compartment or position in the cell). A fluorescent signal is acquired, which corresponds to the degree of interaction that occurred between the partner proteins, which usually follows a stimulus that induces proper translocation. Such assay systems can be used to screen for compounds or treatments that modify either the translocation process involved or the interaction between target proteins used to construct complementary probes.

  β-catenin is a multifunctional protein and plays an important role in the development and progression of certain human cancers (most notably colon cancer). In non-dividing colonic epithelial cells, β-catenin is found in complexes with cell adhesion molecules at the plasma membrane and also controls its targeted destruction (by the cell's ubiquitin-binding system). It is also found in complexes with these proteins (Axin, colorectal adenomatous polyposis protein, or APC, and protein kinase GSK3β). Β-catenin functions as a transcriptional activator. In terms of its function, β-catenin can translocate to the nucleus and form dimers with other pro-transcriptional cofactors (the most prominent in colon cells is TCF4). is necessary. Β-catenin levels in non-dividing cells are kept low by ubiquitin binding and subsequent destruction by proteosomes, and some β-catenin-inducible genes because some β-catenin is found or not found in the nucleus Transcription has occurred or has not occurred. In cancerous and certain precancerous colorectal epithelial cells, mutations in β-catenin itself or in one of its regulatory proteins induce accumulation of the protein in the cytoplasm, followed by translocation to the nucleus and β Catenin: Concomitantly occurs with constitutive activation of genes controlled by TCF4 factor. It is the continuous expression of these genes that induces a cancerous phenotype in such cells.

  In this example, the complementary half of GFP is fused separately with β-catenin and histone H2B linked to the β-catenin binding fragment of transcriptional co-activator TCF4. Both constructs are then cotransfected and expressed in mammalian cells. The β-catenin construct is expected to be localized at low levels throughout the cytoplasm of the cell, and its concentration is effectively controlled by activation of the protein kinase GSK3β. The H2B-TCF4 construct is localized in the nucleus of the cell by a highly basic histone moiety. Therefore, the two components should not meet each other under normal growth conditions. However, when GSK3β is inhibited by lithium ions, it is expected that the cytoplasmic level of the β-catenin construct will increase in the cytoplasm and translocate to the nucleus in a significant fashion. When the construct is mixed together in the nucleus as a result of β-catenin translocation, the natural interaction between the two proteins (β-catenin and TCF4) brings the complementary GFP halves into close proximity, which In the meantime, the annealing process should be initiated, which ultimately induces the generation of a fully fluorescent GFP molecule. Generation of nuclear fluorescence in lithium-treated cells effectively reports integrated results of β-catenin translocation to the nucleus and subsequent interaction between β-catenin and the β-catenin binding domain of TCF4 .

The following fusion constructs were created (see Example 12):
NtermE [F64L] YFP172-β-catenin = plasmid code ps1706
H2B-TCF4 (frag) -CtermEYFP173 = ps1810
Plasmids ps1706 and ps1810 were cotransfected into Chinese hamster ovary cells (CHO) using the transfection agent FuGENE ^™ 6 (Boehringer Mannheim Corp, USA) (according to the method recommended by the supplier). Cells in growth medium (with HAM's F12 nutrient mixture, Glutamax-1, 10% fetal bovine serum (FBS), 100 μg penicillin-streptomycin mixture ml ⁻¹ (supplied by GibcoBRL, Life Technologies, Denmark) In culture. The transfected cells were cultured in this medium in a medium supplemented with two selective agents (1 mg / ml zeocin plus 0.5 mg / ml G418 sulfate) appropriate for the plasmid used. Cells were cultured at 37 ° C. with 100% humidity and normal atmosphere supplemented with 5% CO ₂ .

After culturing for 10-12 days in the continuous presence of the selective agent, it was determined whether the resulting cell line was stably transfected.
For fluorescence microscopy, aliquots of cells are transferred to Lab-Tek chambered cover glasses (Nalge Nunc International, Naperville USA) and allowed to attach for at least about 24 hours until they reach about 80% confluence. Oita. Images were collected using a Zeiss LSM410 inverted confocal fluorescence microscope (Zeiss, Jena, Germany) with a x40 (oil immersion) objective. A 488 nm laser light was used for GFP excitation, and the radiation was filtered through FT510 dichroism and a 510-525 nm bandpass filter.

  After treatment (but before microscopic observation), the cells were fixed as usual with 4% formaldehyde in phosphate buffered saline (PBS) for 10 minutes, and then washed three times with PBS. Carried out.

  FIG. 18 (A) shows the level of fluorescence before applying any treatment to the cells in stable CHO [ps1706 + ps1810]. The level of fluorescence is very weak in many cells, which will be completely dependent on the cell autofluorescence from flavonoids (eg FAD) (the excitation filter used is part of the FAD autofluorescence). In general). Occasionally cells (Occasional cells) (upper right corner of FIG. 18A) show weak GFP fluorescence in the nuclear and cytoplasmic compartments, which is clearly a result of spontaneous complementation. Many of these cells are clustered, possibly inaccurate processing of the introduced components such that they are in close proximity to the same compartment where complementation can occur during expression (or perhaps cell division) It points out the behavior of the clone that would lead to However, when treated with 5 mM LiCl for 16 hours, the number of cells containing fluorescent GFP was greatly increased and many of them had GFP fluorescence restricted exclusively to their nuclei, resulting in this result. Was consistent with the distribution of GFP complementation expected from this pair of fusion constructs (FIG. 18 (B)).

  These results point to the following: using the GFP complementation method, using fusions to the protein components NtermE [F64L] YFP172 and CtermEYFP173, which are normally present in isolated cell compartments or locations, Protein-protein interactions between these components can be monitored, which is the result of a translocation process that brings both components in one compartment or close to one shared position. Based on this principle, a simple intensity or image-based fluorescence assay is used to relate the translocation process associated with a particular cellular component or the interaction between such protein components (live mammalian cells Compounds that affect any of the Use in combination with a similar assay designed to report only the translocation process involved (eg, an assay similar to that described in Example 14 or Example 16), making any compound specific It is possible to define whether to target translocation or interaction.

Example 14: Protein translocation assay using GFP complementation
This method demonstrates the usefulness of the method in screening in vivo for compounds (which are compounds that modify the translocation behavior of the target protein) (translocation dependent complementarity). , TDCzip).

  This example demonstrates that a GFP complementation system can be designed to report target protein translocation behavior in living cells. A fluorescent signal is acquired, which directly corresponds to the degree of translocation that occurs. Such assay systems can be used to screen for compounds or treatments that modify the translocation behavior of the target protein.

  β-catenin is a multifunctional protein and plays an important role in the development and progression of certain human cancers (most notably colon cancer). In non-dividing colonic epithelial cells, β-catenin is found in complexes with cell adhesion molecules at the plasma membrane and also controls its targeted destruction (by the cell's ubiquitin-binding system). It is also found in complexes with these proteins (Axin, colorectal adenomatous polyposis protein, or APC, and protein kinase GSK3β). Β-catenin functions as a transcriptional activator. In terms of its function, β-catenin can translocate to the nucleus and form dimers with other pro-transcriptional cofactors (the most prominent in colon cells is TCF4). is necessary. Β-catenin levels in non-dividing cells are kept low by ubiquitin binding and subsequent destruction by proteosomes, and some β-catenin-inducible genes because some β-catenin is found or not found in the nucleus Transcription has occurred or has not occurred. In cancerous and certain precancerous colorectal epithelial cells, mutations in β-catenin itself or in one of its regulatory proteins induce accumulation of the protein in the cytoplasm, followed by translocation to the nucleus and β Catenin: Concomitantly occurs with constitutive activation of genes controlled by TCF4 factor. It is the continuous expression of these genes that induces a cancerous phenotype in such cells.

  In this example, the complementary half of GFP is fused separately with β-catenin and histone H2B and co-expressed in the cell. The β-catenin construct is expected to be localized at low levels throughout the cytoplasm of the cell, and its concentration is effectively controlled by activation of the protein kinase GSK3β. The H2B construct is localized in the nucleus of the cell by a highly basic histone moiety. Therefore, the two components should not meet each other under normal growth conditions. However, when GSK3β is inhibited by lithium ions, it is expected that the cytoplasmic level of the β-catenin construct will increase in the cytoplasm and translocate to the nucleus in a significant fashion. Both constructs were designed to contain a leucine zipper domain. When the construct is mixed together in the nucleus as a result of β-catenin translocation, the leucine zipper dimerizes to bring the complementary GFP halves into close proximity, during which the annealing process begins, Ultimately induces the generation of a fully fluorescent GFP molecule. Nuclear fluorescence generation in lithium-treated cells effectively reports translocation of β-catenin to the nucleus

The following fusion constructs were created (see Example 12):
NtermE [F64L] YFP172-Zip-H2B = plasmid code ps1736
β-catenin-Zip-NtermE [F64L] YFP172 = ps1758

Plasmids ps1736 and ps1758 were cotransfected into Chinese hamster ovary cells (CHO) using the transfection agent FuGENE ^™ 6 (Boehringer Mannheim Corp, USA) (according to the method recommended by the supplier). Cells in growth medium (with HAM's F12 nutrient mixture, Glutamax-1, 10% fetal bovine serum (FBS), 100 μg penicillin-streptomycin mixture ml ⁻¹ (supplied by GibcoBRL, Life Technologies, Denmark) In culture. The transfected cells were cultured in this medium in a medium supplemented with two selective agents (1 mg / ml zeocin plus 0.5 mg / ml G418 sulfate) appropriate for the plasmid used. Cells were cultured at 37 ° C. with 100% humidity and normal atmosphere supplemented with 5% CO ₂ .

  After culturing for 10-12 days in the continuous presence of the selective agent, it was determined whether the resulting cell line was stably transfected. For fluorescence microscopy, aliquots of cells are transferred to Lab-Tek chambered cover glasses (Nalge Nunc International, Naperville USA) and allowed to attach for at least about 24 hours until they reach about 80% confluence. Oita. Images are collected as specified using a Nikon Diaphot 300 inverted fluorescent microscope (Nikon Corp., Tokyo, Japan), x20 (dry) and / or x40 (oil-immersed) objectives, and Orca ER charged coupler (CCD) camera (Hammamatsu Photonics KK, Hammamatsu City, Japan). Cells were illuminated with a 100 W HBO arc lamp, via a 470 ± 20 nm excitation filter, a 510 nm dichroic mirror and a 515 ± 15 nm emission filter (for minimal image background). Image collection, subsequent fluorescence intensity measurement and analysis were all controlled by specific software (IPLab Spectrum for Windows software, Scanatics, Fairfax, VA USA).

  After treatment, the cells were fixed for 10 minutes with 4% formaldehyde in phosphate buffered saline (PBS) as specified, and then three washing steps with PBS were performed.

  FIG. 19 (A) shows the level of fluorescence before performing any treatment on the cells in stable CHO [ps1736 + ps1758]. The level of fluorescence is very weak, which may be entirely dependent on cellular autofluorescence from flavonoids (eg, FAD) (excitation filters used generally account for some of the FAD autofluorescence). Pass through). In addition, this fluorescence distribution is consistent with FAD autofluorescence, which is mainly present in the cytoplasm, and appears more like dots. When treated with 5 mM LiCl for 16 hours, these cells exhibited GFP-shining nuclei, a result consistent with the distribution of GFP complementation expected from this pair of fusion constructs (FIG. 19 (B ))

  These results point to the following: using the GFP complementation method, using fusions to the protein components NtermE [F64L] YFP172 and CtermEYFP173, which are normally present in isolated cell compartments or locations, The translocation process can be monitored such that both components result in proximity to a one-one compartment or one-one shared location. A simple intensity- or image-based fluorescence assay based on this principle is used to affect the translocation process (in a living mammalian cell) associated with a particular cellular component, Compounds can be screened.

Example 15: Development of inducible FRBP12: FRB interaction
As described in Example 14, some assays experience a sink effect, ie, due to the natural shuttling of any naturally translocating protein, the anchored zipper construct is a constant binding partner. There may be problems with exposure to the supply, which results in irreversible binding and premature fluorophore formation. The same problem, which may be encountered in TIDC systems, relies on constitutive natural interactions (similar to the interaction between β-catenin and TCF-4) (Example 14).

  The FKBP12: FRB system can, for example, reciprocate a nuclear fraction of a translocating protein (fused to complementation partner A) to an anchor protein (fused to complementation partner B) at any particular time. It is a simple means to act.

  The fusion that seems to be most suitable is based on the co-crystal structure of FKBP12: rapamycin: FRB (FIG. 17), which fused both split EYFP F64L fragments to the C-terminus of FKBP12 and FRB. Because they are close to each other. However, this orientation was selected in Example 16 because we generally prefer to fuse the N-terminal split fragment with the fusion partner and visa versa. This is based in part on our experience, and a distance of at least 40 cm between the ends does not limit complementarity (GFP is about 40 cm in length). When using FKBP12 and FRB, a 10-15 residue linker is required on both sides of FKBP12 and FRB. The linker length is between 1.5 Å (α helix) and 3.5 Å (β sheet) per residue (see Figure 17).

Example 16: Protein translocation assay with inducible interaction partners
This example demonstrates the construction of a protein translocation assay using an inducible GFP complementation method, thereby usefulness of the method in screening in vivo for compounds that modify the translocation behavior of the target protein. Is demonstrated (translocation dependent complementation by rapamycin, TIDC).

  This example demonstrates that an inducible GFP complementation system can be designed to report target protein translocation behavior in living cells with an improved signal-to-background ratio. . Following induction of the translocation process, the complementarity interacting proteins are stimulated by proximity to obtain a fluorescent signal (which directly corresponds to the degree of translocation that has occurred). Such an assay system can be used to screen for compounds or treatments that modify the translocation behavior of the target protein.

  Additional linker sequences were added to each. Because we no longer have the relevant closely interacting zipper peptides. The Gateway recombination sequence is about 10 residues, which should be sufficient between the inserted fusion protein and FKBP12 / FRB. The connections for the fragment to be split should be 12-20 residues.

  β-catenin is a multifunctional protein and plays an important role in the development and progression of certain human cancers (most notably colon cancer). In non-dividing colonic epithelial cells, β-catenin is found in complexes with cell adhesion molecules at the plasma membrane and also controls its targeted destruction (by the cell's ubiquitin-binding system). It is also found in complexes with these proteins (Axin, colorectal adenomatous polyposis protein, or APC, and protein kinase GSK3β). Β-catenin functions as a transcriptional activator. In terms of its function, β-catenin can translocate to the nucleus and form dimers with other pro-transcriptional cofactors (the most prominent in colon cells is TCF4). is necessary. Β-catenin levels in non-dividing cells are kept low by ubiquitin binding and subsequent destruction by proteosomes, and some β-catenin-inducible genes because some β-catenin is found or not found in the nucleus Transcription has occurred or has not occurred. In cancerous and certain precancerous colorectal epithelial cells, mutations in β-catenin itself or in one of its regulatory proteins induce accumulation of the protein in the cytoplasm, followed by translocation to the nucleus and β Catenin: Concomitantly occurs with constitutive activation of genes controlled by TCF4 factor. It is the continuous expression of these genes that induces a cancerous phenotype in such cells.

  In this example, the complementary half of GFP is fused separately with β-catenin and histone H2B and co-expressed in the cell. The β-catenin construct is expected to be localized at low levels throughout the cytoplasm of the cell, and its concentration is effectively controlled by activation of the protein kinase GSK3β. The H2B construct is localized in the nucleus of the cell by a highly basic histone moiety. Therefore, the two components should not meet each other under normal growth conditions. However, when GSK3β is inhibited by lithium ions, it is expected that the cytoplasmic level of the β-catenin construct will increase in the cytoplasm and translocate to the nucleus in a significant fashion. Both constructs were designed to contain inducible interaction domains. The β-catenin construct contains FK506 binding protein (FKBP). The H2B construct includes FRB (which is the 93 amino acid portion of FRAP), the large Pl3 kinase homolog FRAP (also known as mTOR or RAFT). Immunosuppressive compound Rapamycin binds FKBP and simultaneously FRB and acts as a heterodimerizer for these two proteins. Complementary reactions between the separated halves of GFP (contained in these two constructs) can be induced by the addition of rapamycin, but only if the protein constructs can meet each other at the same cellular location. Since FKBP and FRB do not interact in the absence of rapamycin, the “sink effect” is greatly reduced or eliminated.

  When the construct is mixed together in the nucleus as a result of β-catenin translocation, the addition of rapamycin brings the complementary GFP halves into close proximity via an inducible FKBP: FRB interaction, An annealing process should be initiated, which ultimately induces the generation of a fully fluorescent GFP molecule. In this example, the occurrence of nuclear GFP fluorescence (which occurs after complementation) is induced by rapamycin, resulting in a report of the extent of translocation of the β-catenin construct to the nucleus upon addition of rapamycin.

The following fusion constructs were created (see Example 12):
NtermE [F64L] YFP172-FKBP-βcatenin = plasmid code ps1809
H2B-FRAP (2024-2114) -CtermEYFP173 = ps1827

Plasmids ps1809 and ps1827 were cotransfected into Chinese hamster ovary cells (CHO) using the transfection agent FuGENE ^™ 6 (Boehringer Mannheim Corp, USA) (according to the method recommended by the supplier). Cells in growth medium (with HAM's F12 nutrient mixture, Glutamax-1, 10% fetal bovine serum (FBS), 100 μg penicillin-streptomycin mixture ml ⁻¹ (supplied by GibcoBRL, Life Technologies, Denmark) In culture. The transfected cells were cultured in a medium supplemented with two selective agents (1 mg / ml zeocin plus 0.5 mg / ml G418 sulfate) appropriate for the plasmid used in this medium. Cells were cultured at 37 ° C. with 100% humidity and normal atmosphere supplemented with 5% CO ₂ .

  After culturing for 10-12 days in the continuous presence of the selective agent, it was determined whether the resulting cell line was stably transfected. For fluorescence microscopy, aliquots of cells are transferred to Lab-Tek chambered cover glasses (Nalge Nunc International, Naperville USA) and allowed to attach for at least about 24 hours until they reach about 80% confluence. Oita. All treatments were performed in full HAM's medium + 10% FBS.

  Images were collected using a Zeiss LSM410 inverted confocal fluorescence microscope (Zeiss, Jena, Germany) with a x40 (oil immersion) objective. A 488 nm laser light was used to excite GFP and the emissions were filtered through FT510 dichroism and a 510-525 nm bandpass filter.

  After treatment (but before microscopic observation), the cells were fixed as usual with 4% formaldehyde in phosphate buffered saline (PBS) for 10 minutes, and then washed three times with PBS. Carried out.

  FIG. 20 shows a montage of CHO cells (expressing ps1809 and ps1827), which was imaged from four separate treatments. The top row (FIGS. 20a-d) is an image of GFP fluorescence. GFP is localized only in the nucleus of these cells. This is as demonstrated in the lower row image (FIGS. 20e-h) where the passed light image is overlaid with the corresponding GFP fluorescence. In FIGS. 20c & d, immediately after transfer to Lab-Tek chambered coverglass, treatment of cells with 5 mM lithium chloride for 16 hours inhibits kinase GSK3; cells in FIGS. Did not receive lithium. The cells of FIGS. 20b & d were then treated with 3 μM rapamycin (the final ethanol concentration in the ethanol stock was 0.6% v / v), whereas the cells of FIGS. 20a & c were only 0.6% ethanol. Was processed. Both treatments were continued for an additional 6 hours (before fixing and imaging the cells).

  Without lithium treatment (FIGS. 20a & b), the level of fluorescence was very weak in many cells, which would be entirely dependent on cellular autofluorescence from flavonoids (eg FAD) (excitation used) The filter generally allows some of the FAD autofluorescence to pass through).

  Occasionally cells (Occasional cells) show weak GFP fluorescence in the nucleus, which is clearly a result of spontaneous complementation that would accompany nuclear membrane disruption during cell division. When treated with 5 mM LiCl for 16 hours, the number of cells containing fluorescent GFP was greatly increased, many of them having GFP fluorescence limited exclusively to their nuclei, and this result is It was consistent with the distribution of GFP complementation expected from the paired fusion constructs (FIGS. 20c & d). The level of nuclear fluorescence in Li-treated cells is substantially increased with rapamycin treatment (FIG. 20d), and the dimerization factor is a cell with a bright nucleus in each responding cell. Increase both the number and the average intensity of GFP.

  In the screening situation, this example shows that (a) spontaneous complementarity does not occur in untreated cells (compared to FIG. 19A), thereby avoiding the “sink” effect. .

  These results point to the following: it is possible to monitor the translocation of protein components in living cells by configuring the GFP complementation method and the efficiency of the method Can be significantly improved by including an inducible protein-protein interaction link (eg, provided by FKBP: FRB in the presence of rapamycin) (as measured by signal background ratio). Be possible. Using such a system configuration, a high-throughput, intensity-based fluorescence assay can be constructed to identify compounds (or other influences) that act to modulate the translocation of cellular components. It will be possible.

  table

Table 2 Oligonucleotides used for cloning. Oligonucleotides beginning with P * are phosphorylated at the 5 ′ end to allow ligation.

Table 3 Primer pairs used for EGFP fragment amplification

Table 4 Cloning and expression vectors

Table 5 Sequence names and numbers

  All cited patents, literature, co-pending applications, and provisional applications referenced in this application are hereby incorporated by reference.

  It will be apparent that the described invention can be varied in many ways. Such changes are not recognized as departing from the spirit and scope of the present invention. Also, all such modifications as would be obvious to one skilled in the art are included within the scope of the claims appended hereto.

General structure of the fusion protein coding sequence. 16-bit image of fluorescent CHO-hIR cells co-transfected with NtermEGFP-NZ and CZ-CtermEGFP expression vectors or transfected with pEGFP-C1. The images are acquired individually and scaled to visualize the cells and the fluorescence distribution therein. Due to pixel intensity scaling, relative fluorescence levels are not comparable between images. The split site exists either between residues 157/158 (top row, plasmids PS1557 and PS1559) or between residues 172/173 (center row, plasmids PS1558 and PS1560). The EGFP expression vector pEGFP-C1 was transfected into the cells in the bottom row. Images were acquired on day 1 (left column), day 2 (center column), or day 10 (right column) after transfection. Cell images are representative of cells that express functionally complementary fragments. 16-bit image of fluorescent CHO-hIR cells co-transfected with NtermEGFP-NZ and CZ-CtermEGFP expression vectors or transfected with pEGFP-C1. The image is similar to that shown in FIG. 2, but shown in FIG. 3 with the same intensity scaling to allow comparison of fluorescence intensity. Cells transfected with a complementation construct based on the split between residues 172 and 173 (middle row) are more apparent than cells transfected with a complementation construct based on the split between residues 157 and 158 (top row) Due to strong fluorescence. However, cells transfected with the pEGFP-C1 construct (bottom row) exhibit significantly stronger fluorescence on day 2. The raw microscope image shown in FIG. 3 was analyzed using the ImageJ software package, and data analysis was performed with Microsoft Excel. For each 16-bit monochromatic IP Lab microscope image, pixel intensity data was generated with ImageJ and exported to an Excel spreadsheet for data analysis. The darkest and brighttest 0.5% pixels were identified in each image and the average intensity of these two groups of pixels was calculated. The average intensity of the darkest pixel at 0.5% was defined as the background fluorescence intensity (shown in the histogram as a white bar) and the intensity of the brightest pixel at 0.5% was defined as the maximum intensity. The difference in intensity between maximum intensity and background intensity was defined as the response (shown in the histogram as a cross hatch bar). The sum of background intensity and response is equal to the maximum intensity. From the figure it is clear that it is EGFP-based fluorescence complementation with residues 157 and 173 between residues 172 and 173 (and possibly anywhere else in this loop) It is very good for fluorescence complementarity based on EGFP using splitting between 158 (and possibly splitting in another region within this loop). Appropriate fluorescent protein cleavage sites are indicated by a GFP ribbon and wireframe representation. The two representations show the same site from two planes (molecules rotated about 180 degrees around the vertical axis). Co-transfection of expression vectors expressing complementary fragments based on EGFP and EYFP. As described in FIG. 3, to compare the ability of various complementary fragments, they are combined in a cell to produce a functional complex. All images are similarly scaled, allowing a direct comparison of fluorescence intensity between images. A single transfection with an N-terminal fragment did not produce any detectable fluorescence above background levels (data not shown). These N-terminal fragments contain amino acid residues 65-67 that form a chromophore in full-length GFP. Quantitative analysis of the image shown in FIG. The results are consistent with the impression of visual inspection of the cells. Data was obtained as described in the legend of FIG. Co-transfection of expression vectors expressing complementary fragments based on EGFP and EYFP. To compare the effect of mixing EGFP, EYFP and EYFP F64L fragments of different colors and to overlap the fragments (eg, encoding residues 1-172 and 158-238 as described in FIG. 3) To determine the effect of combining the fragments). All color combinations are complementary, but generally less efficient than the correct combination. Fragments with overlapping regions are also functional, which may be advantageous in experiments where longer linker sequences are required (or will be required) by the fusion partner due to steric hindrance. This is not the case in this experiment where the fusion partner is a leucine zipper. In the example (middle column), residues 158-172 were present in both fragments. In all situations, F64L has a positive effect on fluorescence intensity. All images are similarly scaled, allowing a direct comparison of fluorescence intensity between images. Quantitative analysis of the image shown in FIG. The results can be directly compared with the results shown in FIG. Also, they are consistent with the visual search impression of the cells. Data was obtained as described in the legend of FIG. CHO-hIR [PS1767 + PS1768] cells at 3 time points after treatment with 1 μM rapamycin. Image (c) was acquired with an exposure of 25 msec, and the previous two images were acquired with an exposure of 100 msec. (A) is the starting condition for these cells (t = 0) and the fluorescence is barely visible in most cells, but nevertheless some cells in the population (<5% However, it was noted that it had low (but appreciable) fluorescence before treatment. (B) After 4 hours, many cells (about 40%) generated significantly higher EYFP fluorescence throughout the cytoplasm and nucleus location. (C) After 16 hours, the cell-by-cell response increased further and encompassed a large proportion (about 70%) of the cell population. Rate of generation of cellular EYFP fluorescence after rapamycin treatment of CHO-hIR [PS1767 + PS1768] strain. Cells were treated with 3 μM rapamycin in 96 well plates and fluorescence was measured. Treatments and measurements were performed on cells grown in Ham's medium + 10% FBS and fluorescence measurements were corrected for background fluorescence from this medium. The graph demonstrates that the half-time for fluorescence generation is about 5 hours. Values corrected for HAM's background, each value is the average of 8 measurements + sd. Response curves of different rapamycin doses to the CHO-hIR [PS1769 + PS1771] cell line. Cells were cultured in 96-well plates, treated with various rapamycin doses for 16 hours, then fixed and stained with Hoechst before EYFP fluorescence / cells (arbitrary units) were determined on an Ascent plate reader. Values are corrected for PBS background and cell number. The cell line shows an approximately 3-fold increase in EYFP intensity / cell over the rapamycin dose range used in this experiment. Each cell line was fluorescently activated and sorted into three groups: (i) the most green group (ii) moderate to low green group and (iii) black group . The “most green” was discarded in each case, while the other two groups were cultured for further use. A: CHO-hIR [ps1768 + ps1767] FACS group “black” before stimulation (i) and after stimulation with 100 nM rapamycin (ii) & (iii) for 16 hours. Images (i) and (ii) were exposed for 100 msec and image (iii) was exposed for 25 msec. B: CHO-hIR [ps1768 + ps1767] FACS group “moderate to low green” before stimulation (i) and after stimulation with 100 nM rapamycin (ii) & (iii) for 16 hours. Images (i) and (ii) were exposed for 100 msec and image (iii) was exposed for 25 msec. The responses of the FACS groups (respectively) of “moderate to low green” (a) and “black” (b) derived from the CHO-hIR [PS1767 + PS1768] parent strain are shown. The dose response to rapamycin was measured after 7 hours (i) and 30 hours (ii) for each cell line. Values for fluorescence were corrected for plate & media background. The increase in EYFP fluorescence is over 20 times the unstimulated value in each case. Dose response in two FACS'd strains, (a) CHO-hIR [PS1768 + PS1767] “moderate to low green” group, and (b) CHO-hIR [PS1769 + PS1771] “black” group for FK506 vs. 100 nM rapamycin A competition curve is shown. The EC ₅₀ value for both cases is approximately 1.2 μM FK506. Cells were incubated overnight (16 hours) with a mixture of the two compounds, then fixed and stained with Hoechst on an Ascent plate reader prior to EYFP fluorescence / cell determination. Plate and solution backgrounds are subtracted; the dashed lines on each graph indicate the level of fluorescence before stimulation for each cell line in these experiments. Fluorescent protein alignment Fluorescent protein alignment Crystal structure of rapamycin-induced binding of FRBP12 (left) to FRB (right). The C-termini of the molecules are within 15 cm of each other. The N-terminus of one molecule is 32-36 cm away from the C-terminus of the other molecule. (A) shows the level of fluorescence before the cells are subjected to any treatment in stable CHO [ps1706 + ps1810] (NtermE [F64L] YFP172-βcatenin + H2B-TCF4 (frag) -CtermEYFP173). The level of fluorescence is very weak in many cells, which will be completely dependent on the cell autofluorescence from flavonoids (eg FAD) (the excitation filter used is part of the FAD autofluorescence). In general). Occasionally cells (Occasional upper right corner) show weak GFP fluorescence in the nucleus and cytoplasmic position, which is clearly a result of spontaneous complementation. Many of these cells are clustered, possibly inaccurate processing of the introduced components such that they are close to the same location where complementation can occur during expression (or perhaps cell division). It points out the behavior of the clone that would lead to However, when treated with 5 mM LiCl for 16 hours, the number of cells containing fluorescent GFP was greatly increased and many of them had GFP fluorescence restricted exclusively to their nuclei, resulting in this result. Was consistent with the distribution of GFP complementation expected from this pair of fusion constructs (B). (A) shows the level of fluorescence before performing any treatment on the cells in stable CHO [ps1736 + ps1758] (NtermE [F64L] YFP172-Zip-H2B + β-catenin-Zip-CtermEYFP173). When treated with 5 mM LiCl for 16 hours, these cells exhibited GFP-shining nuclei, a result consistent with the distribution of GFP complementation expected from this pair of fusion constructs (B). A montage of CHO cells (NtermE [F64L] YFP172-FKBP-β-catenin + H2B-FRAP (2024-2114) -CtermEYFP173) expressing ps1809 and ps1827 was imaged from four separate treatments. Is. The top row (ad) is an image of GFP fluorescence. The lower row image (eh) overlays the passed light image with the corresponding GFP fluorescence. a & e: Unprocessed. b & f: 3 μM rapamycin (+6 hours). c & g: 16 mM treatment with 5 mM lithium chloride. d & h: 16 hours treated with 5 mM lithium chloride 3 μM rapamycin (+6 hours). Various assay configurations. IDC (interaction-dependent complementarity) is a normal configuration. In this configuration, the interaction between the two proteins (denoted here BetaCat and Tcf4) acts on the N and C-terminal XFP complementation partners to allow reconstitution and detection. One particular advantage of an IDC pair is that it validates a TIDC assay generated from a selected protein pair. A rapamycin-inducible FKBP-FRB interaction pair can be used to confirm and further develop the use of IDC. See also the description of the TDCrap system. TIDC (translocation and interaction-dependent complementarity) is a configuration in which redistribution and interaction are measured as an increase in light intensity. Β-catenin fused to the complementary fragment is preferentially located in the cytoplasm. Upon stimulation, β-catenin translocates to the nucleus. Here it finds the natural interaction partner TCF-4 (fused with a complementary fragment). The two complementarity partners associate and become fluorescent. Compounds that inhibit either β-catenin translocation or β-catenin-TCF-4 interaction inhibit complementation and the formation of fluorescent complexes. TDC (translocation dependent complementarity) is a configuration in which only the redistribution is measured as an increase in light intensity. For example, β-catenin fused with an XFP complementary fragment and a leucine zipper sequence is preferentially located in the cytoplasm. Upon stimulation, the β-catenin fusion translocates to the nucleus. Here, the leucine zipper discovers other leucine zipper sequences, which are fused with complementary XFP fragments and anchor proteins or nuclear localization signals. The two complementarity partners are assembled and become fluorescent due to the interaction of the zipper. Compounds that inhibit β-catenin translocation inhibit complementation and the formation of fluorescent complexes. TDCrap (translocation-dependent complementarity induced by rapamycin) is essentially the same as TDC, but inducible interaction pairs (eg, rapamycin-induced FKBP and FRB interaction pairs) are: The “sink effect” is greatly reduced, facilitating the development of other types of translocation (eg, cytoplasm to plasma membrane or visa versa). The left hand portion of the figure shows the assay configuration for determining receptor dimerization (in this case, homodimerization). Two conjugates comprising the receptor and the N-terminus of the complementary protein (here YN) or the C-terminus of the complementary protein (here YC) are expressed in the cell. Only upon stimulation of the cell with the ligand, the receptors are in close proximity and the YN and YC terminal fragments form a functional protein. The right hand side of the figure illustrates the configuration of the assay for caspase activity. The first conjugate consists of the complementary protein N-terminus (YN), constitutive interaction partner (leucine zipper), nuclear localization sequence (NLS), caspase consensus cleavage site (eg, VAD = valine-alanine- Aspartate) and fused to an anchor protein (here, cell membrane). The second conjugate comprises the N-terminus (YC) of the complementary protein, the interaction partner (leucine zipper) and the anchor in the nucleus (shown here as a circle). When caspase activity is increased, the caspase cleaves the YN fusion at the VAD site and the first conjugate translocates to the nucleus by NLS, where fluorescence occurs upon binding to an interaction partner (zipper).

Claims (75)

A cell comprising at least:
A first conjugate comprising an N-terminal fragment of the first protein and a complementary protein; and
A second conjugate comprising a C-terminal fragment of a second protein and a complementary protein;
Here, the first conjugate has a preferential position at a different cell position from the position where the second conjugate is preferentially located,
Wherein the first protein and the second protein bind to each other,
Here, the complementary protein changes when the two fragments of the complementary protein move to a close position and the two fragments of the complementary protein form a fully functional protein ( altered characteristics). The cell according to claim 1, wherein the cell compartment of the first conjugate is determined by the nature of the first protein. The cell according to claim 1 or 2, wherein the cell compartment of the second conjugate is determined by the nature of the second protein. 3. The cell according to claim 1, wherein the second conjugate further comprises an anchor protein, and the anchor protein is different from the position where the first protein is preferentially located. A cell that anchors to a compartment. A cell comprising at least:
A first conjugate comprising a first protein, an interaction partner A and a first terminal fragment of a complementary protein; and
A second conjugate comprising an interaction partner B and a second terminal fragment of the complementary protein;
Wherein the first conjugate has a preferential position in a cell compartment different from the position where the second conjugate is preferentially located;
Here, the complementary protein changes when the two fragments of the complementary protein move to a close position and the two fragments of the complementary protein form a fully functional protein ( altered characteristics). 6. The cell according to claim 5, wherein the interaction partner A and interaction partner B bind to each other. 6. The cell of claim 5, wherein the interaction partner A and interaction partner B bind to each other only when an interaction stimulus is applied. The cell according to any one of claims 5 to 7, wherein the second conjugate further comprises an anchor protein, and the anchor protein is different from a position where the first protein is preferentially located. A cell that anchors to a compartment. The cell according to any one of claims 1 to 8, wherein the complementary protein is dihydrofolate reductase (DHFR). The cell according to any one of claims 1 to 8, wherein the complementary protein is ubiquitin. The cell according to any one of claims 1 to 8, wherein the complementary protein is β-lactamase. The cell according to any one of claims 1 to 8, wherein the complementary protein is β-galactosidase. The cell according to any one of claims 1 to 8, wherein the complementary protein is a fluorescent protein. 14. The cell according to claim 13, wherein the complementary protein is green fluorescent protein (GFP). 15. The cell according to claim 14, wherein the complementary protein is derived from Aequoria victoria . 15. The cell of claim 14, wherein the N-terminal fragment of the complementary protein is an N-terminal fragment of GFP (comprising a continuous stretch of amino acids from GFP amino acid number 1 to amino acid number X), Here, the peptide bond between amino acid number X and amino acid number X + 1 is in the loop of GFP, and the C-terminal fragment of the complementary protein is the C-terminal fragment of GFP (of amino acids from amino acid number X + 1 to amino acid number 238 of GFP). A cell comprising a continuous stretch). 15. The cell of claim 14, wherein the N-terminal fragment of the complementary protein is an N-terminal fragment of GFP (comprising a continuous stretch of amino acids from GFP amino acid number 1 to amino acid number X), Here, the peptide bond between amino acid number X and amino acid number X + 1 is in the loop of GFP, and the C-terminus of the complementary protein is the C-terminal fragment of GFP (a sequence of amino acids from amino acid number Y + 1 to amino acid number 238 of GFP). Where Y <X results in the overlap of two GFP fragments and the peptide bond between amino acid Y and amino acid Y + 1 is within the loop of GFP. The cell according to any one of claims 14 to 17, wherein the GFP is selected from the group consisting of EGFP, EYFP, ECFP, dsRed, and Renilla GFP. The cell according to claim 18, wherein the GFP is EGFP. The cell according to claim 18, wherein the GFP is EYFP. 21. The cell according to any one of claims 14 to 20, wherein the amino acid at position 1 preceding the chromophore is mutated to provide an increase in fluorescence intensity. The cell according to claim 21, wherein the amino acid F at position 1 preceding the chromophore is substituted with L. 23. The cell according to any one of claims 14 to 22, wherein the GFP is mutated and further has a S72A mutation. 24. The cell of any one of claims 16-23, wherein X is between 9 and 10 in the Thr9-Val11 loop; or between 23 and 24 in the Asn23-His25 loop; or Thr38 ~ Between 38 and 39 in the Gly40 loop; or 48 and 55 in the Cys48-Pro56 loop; or 72 and 75 in the Ser72-Asp76 loop; or 81 and 82 in the His81-Phe83 loop. Or between 88 and 89 in the Met88-Glu90 loop; between 101 and 102 in the Lys101-Asp103 loop; or between 114 and 117 in the Phe114-Thr118 loop; or the Ile128-Tyr145 loop Between 128 and 144 in; or between 154 and 159 in the Ala154-Gly160 loop ; Or lle171~Ser175 between 171 and 174 in the loop; cells is between 210 and 214 or Asp210~Art215 loop; between 188 and 196 or lle188~Asp197 the loop. 25. The cell of claim 24, wherein X is between 154 and 159 in the Ala154-Gly160 loop. 26. The cell of claim 25, wherein X is 157 in the Ala154-Gly160 loop. 25. The cell of claim 24, wherein X is between 171 and 174 within the lle171-Ser175 loop. 28. The cell of claim 27, wherein X is 172 within the lle171-Ser175 loop. 18. A cell according to claim 17, wherein Y is between 154 and 159 in the Ala154-Gly160 loop. 30. The cell of claim 29, wherein Y is 157 in the Ala154-Gly160 loop. 31. The cell according to any one of claims 16 to 30, wherein X is 172 in the llle171-Ser175 loop and Y is 157 in the Ala154-Gly160 loop. 32. The cell according to any one of claims 1 to 31, wherein the N-terminal fragment of the complementary protein is fused in frame with the first protein of interest. The cell according to any one of claims 1 to 32, wherein the first protein is fused with the N-terminus of the N-terminal fragment of the complementary protein. 34. The cell according to any one of claims 1 to 33, wherein the first protein is fused with the C-terminus of the N-terminal fragment of the complementary protein. 35. The cell according to any one of claims 1-34, wherein the C-terminal fragment of the complementary protein is fused with the second protein in frame. 36. The cell according to any one of claims 1 to 35, wherein the second protein of interest is fused with the N-terminus of the C-terminal fragment of the complementary protein. 37. The cell according to any one of claims 1-36, wherein the second protein of interest is fused to the C-terminus of the C-terminal fragment of the complementary protein. 38. The cell according to any one of claims 1 to 37, wherein the N-terminal fragment of the complementary protein (fused with the first protein in frame) further comprises a linker sequence of the complementary protein. A cell provided between the N-terminal fragment and the first protein. The cell according to any one of claims 1 to 38, wherein the C-terminal fragment of the complementary protein (fused with the second protein of interest in frame) further comprises a linker sequence, A cell provided between the C-terminal fragment of the protein and the second protein. The cell according to any one of claims 1 to 39, wherein the GFP is EYFP (further having a F64L mutation), X is 172, and the first protein (fused with an N-terminal fragment of GFP) Cells) fused to the C-terminus of the N-terminal fragment of GFP, and the second protein (fused to the C-terminal fragment of GFP) is fused to the N-terminus of the GFP C-terminal fragment. 41. The cell according to any one of claims 1 to 40, wherein the GFP is EYFP (further having an F64L mutation), X is 157, and the first protein (fused with an N-terminal fragment of GFP). Cells) fused to the C-terminus of the N-terminal fragment of GFP, and the second protein (fused to the C-terminal fragment of GFP) is fused to the N-terminus of the GFP C-terminal fragment. 42. The cell according to any one of claims 1-41, wherein the first conjugate is expressed in the cell. 43. The cell according to any one of claims 1-42, wherein the second conjugate is expressed in the cell. 44. A cell according to any one of claims 1 to 43, wherein interaction partner A is FKBP12, interaction partner B is FRAP (or reverse), and said interaction stimulation Are rapamycin cells. 45. A cell according to any one of claims 1 to 44, wherein interaction partner A is FKBP12, interaction partner B is FRB (T2098L) (or vice versa) and said Cells that interact with rapamycin. 46. A cell according to any one of claims 1 to 45, wherein interaction partner A is FKBP12, interaction partner B is FRB (T2098L) (or vice versa) and said Cells whose interaction stimulus is AP21967. 49. A cell according to any one of claims 1 to 46, wherein interaction partner A is FKBP12, interaction partner B is FKBP12 (or vice versa) and said interaction stimulus Is the cell that is AP21967. 48. The cell of any one of claims 1-47, wherein interaction partner A is FKBP12, interaction partner B is FKBP12 (or vice versa) and said interaction stimulus Is the cell that is AP21967. 49. The cell according to any one of claims 1 to 48, wherein the interaction partners A and B are steroid hormone receptors and the dimerizing agent is a cognate hormone ligand. cell. 50. A cell according to any one of claims 1 to 49, wherein the interaction partners A and B are estrogen receptors and the interaction stimulating factor is estrogen. 51. The cell according to any one of claims 1-50, wherein the anchor protein comprises a transmembrane domain of epidermal growth factor receptor (EGFR) or one transmembrane domain of the integrin protein family. Or a protein that contains a myristoylation sequence of c-Src (residues 1-14). 52. The cell according to any one of claims 1 to 51, wherein the anchor protein is a protein normally limited to a histone protein or a nucleolus (eg, a p120 nucleolus protein). 53. The cell according to any one of claims 1 to 52, wherein the anchor protein is a protein that is normally restricted to the outer or inner membrane of the mitochondria (eg, VDAC, Fo subunit of ATP-ase, or NADH dehydrogenase). 54. The cell according to any one of claims 1 to 53, wherein the anchor protein is a protein (eg, TGN38 or ADAM12-L) that is usually limited to various different regions of the Golgi apparatus. 55. The cell according to any one of claims 1 to 54, wherein the anchor protein is a protein (e.g., P125, FAK, integrin alpha or beta) that is normally limited to focal adhesion complexes. Or cells that are paxillin). 56. The cell of any one of claims 1 to 55, wherein the anchor protein is a protein normally associated with a cytoskeletal structure (e.g., F-actin chain or microtubule bundle, e.g., MAP4, alpha-actinin). Actin-binding domain, kinesin, myosin or dynein). 57. The cell according to any one of claims 1 to 56, wherein the anchor protein is a protein normally associated with a nuclear material or a nuclear component [eg, histone proteins (histone H1, H2A, H2B, H3, H4, and variants thereof). 58. The cell according to any one of claims 1 to 57, wherein the anchor protein is a nuclear membrane (for example, A and B type lamin) or a nucleolus (for example, a splicing body, a Kahal body, PML). A cell that is a protein normally associated with the nucleolus (PML carcinogenic domain, PODS), or a transcription engine (eg, RNA polymerase POL-II). 59. The cell according to any one of claims 1 to 58, wherein the cell is a eukaryotic cell. 59. The cell according to any one of claims 1 to 58, wherein the cell is a prokaryotic cell. A method for detecting protein-protein interactions comprising the following steps:
(A) providing a cell comprising at least:
A first conjugate comprising a first terminal fragment of the first protein of interest and a complementary protein; and
A second conjugate comprising a second terminal fragment of said second protein of interest and a complementary protein;
Here, the second protein of interest has a preferential position at a different cell position from the position where the first protein of interest is preferentially located.
(B) determining whether complementation partner A and complementation partner B are complementary;
Complementarity between complementation partner A and complementation partner B is as follows: the subject first protein is located from the preferential position of the subject first protein, and the subject second protein is located. A method that is an indicator of translocation to a cellular compartment and that the two proteins of interest interact. A method for detecting protein-protein interactions comprising the following steps:
(A) providing the cell according to any one of claims 1 to 60; and
(B) determining whether complementation partner A and complementation partner B are complementary;
Complementarity between complementation partner A and complementation partner B is as follows: the subject first protein is located from the preferential position of the subject first protein, and the subject second protein is located. A method that is an indicator of translocation to a cellular compartment and that the two proteins of interest interact. A method for testing whether a compound inhibits a protein-protein interaction comprising the following steps:
(A) providing the cell according to any one of claims 1 to 60;
(A1) adding said compound to the cells of step (a);
(A2) adding a translocation stimulus to the cells of step (a1);
Wherein the translocation stimulus acts on the two conjugates to produce the same cellular localization;
(B) determining whether complementation partner A and complementation partner B are complementary;
A method wherein low complementarity (compared to a system omitting step (a1)) is an indication that the compound has the ability to block the interaction between two proteins. 64. The method according to any one of claims 61 to 63, wherein the second conjugate further comprises an anchor protein, and the anchor protein has a position where the target first protein is preferentially located. Anchors to different cell compartments. A method for detecting translocation of a protein comprising the following steps:
(A) providing a cell comprising at least:
A first conjugate comprising said protein, a first terminal of a complementary protein, and an interaction partner A; and
A second conjugate comprising an anchor protein, a second end of a complementary protein, and an interaction partner B;
Wherein the anchor protein anchors to a cell compartment different from the cell compartment in which the protein is preferentially located; and
Wherein said interaction partner A and interaction partner B bind to each other;
(B) determining whether complementation partner A and complementation partner B are complementary;
Complementarity between complementation partner A and complementation partner B is as follows: the protein is translocated from a preferential position of the protein to a cell compartment to which the anchor protein anchors. The method that is an indicator. A method for detecting translocation of a protein comprising the following steps:
(A) providing a cell according to any of claims 6 to 60; and
(B) determining whether complementation partner A and complementation partner B are complementary;
Complementarity between complementation partner A and complementation partner B is as follows: the protein is translocated from a preferential position of the protein to a cell compartment to which the anchor protein anchors. The method that is an indicator. A method for testing whether a compound induces protein translocation comprising the following steps:
(A) providing the cell according to any one of claims 6 to 60;
(A1) adding said compound to the cells of step (a);
(B) determining whether complementation partner A and complementation partner B are complementary;
A method wherein increased complementarity (compared to a system omitting step (a1)) is an indication that the compound induces translocation of the protein. A method for testing whether a compound inhibits protein translocation comprising the following steps:
(A) providing the cell according to any one of claims 6 to 60;
(A1) adding said compound to the cells of step (a);
(A2) adding a translocation stimulus to the cells of step (a1):
(B) determining whether complementation partner A and complementation partner B are complementary;
A method wherein low complementarity (compared to a system that omits step (a1)) is an indication that the compound blocks translocation of the protein. A method for detecting translocation of a protein comprising the following steps:
(A) providing a cell comprising at least:
A first conjugate comprising said protein, complementation partner A, and interaction partner A; and
A second conjugate comprising an anchor protein, a complementation partner B, and an interaction partner B;
Wherein the anchor protein anchors to a cell compartment different from the cell compartment in which the protein is preferentially located,
Wherein said interaction partner A and interaction partner B bind to each other only when an interaction stimulus is applied;
(B) adding an interaction stimulus;
(C) determining whether complementation partner A and complementation partner B are complementary;
Complementarity between complementation partner A and complementation partner B is as follows: after the addition of the interaction stimulus, the protein is moved from the preferential position of the protein to the cell compartment anchored by the anchor protein. A method that is an indicator of being translocated. A method for detecting translocation of a protein comprising the following steps:
(A) providing the cell according to any one of claims 7 to 60; and
(B) adding an interaction stimulus;
(C) determining whether complementation partner A and complementation partner B are complementary;
Complementarity between complementation partner A and complementation partner B is as follows: after the addition of the interaction stimulus, the protein from the preferential position of the protein to the cell compartment anchored by the anchor protein A method that is an indicator of being translocated. A method for testing whether a compound induces protein translocation comprising the following steps:
(A) providing the cell according to any one of claims 7 to 60;
(A1) adding said compound to the cells of step (a);
(B) adding an interaction stimulus;
(C) determining whether complementation partner A and complementation partner B are complementary;
A method wherein increased complementarity (compared to a system omitting step (a1)) is an indication that the compound induces translocation of the protein. A method for testing whether a compound inhibits protein translocation comprising the following steps:
(A) providing the cell according to any one of claims 7 to 60;
(A1) adding said compound to the cells of step (a);
(A2) adding a translocation stimulus to the cells of step (a1):
(B) adding an interaction stimulus;
(C) determining whether complementation partner A and complementation partner B are complementary;
A method wherein low complementarity (compared to a system that omits step (a1)) is an indication that the compound blocks translocation of the protein. A kit for detecting an antigen comprising:
(A) a first antibody that binds to the antigen;
(B) a first conjugate comprising a protein that binds to the first antibody and the N-terminus of a complementary protein;
(C) a second conjugate comprising a protein that binds to the first antibody and the C-terminus of the complementary protein;
A kit wherein binding of the first and second conjugates to the first antibody brings the two ends of the complementary proteins in proximity such that a functional protein is formed. A method for determining caspase activity in a cell comprising at least the following steps:
(A) providing a cell comprising at least:
A first conjugate comprising a first end of a complementary protein, an interaction partner A, a nuclear localization sequence (NLS), a valine-alanine-aspartate (VAD) sequence and an anchor protein;
A second conjugate comprising a second end of a complementary protein, an interaction partner B and an anchor protein;
Wherein the anchor protein of the first conjugate is present at a different cellular location than the nucleus, and the anchor protein of the second conjugate is present in the nucleus, and
Here, the first ends of the interaction partner A, the NLS and the complementary protein are all located on the same side of the VAD sequence, and the anchor protein is located on the other side of the NLS. ing,
(B) determining whether the complementary proteins are complementary;
A method in which complementarity is an indication of the following: caspase cleaves the first conjugate, the free part translocates to the nucleus, and partner A and complementation partner B complement. A nucleic acid encoding any said conjugate, fragment or fusion protein.

Images