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Composition for detecting protein-protein interactions comprising fragments of secreted alkaline phosphatase (SEAP) and method for detecting protein-protein interactions using the same

20200308559 ยท 2020-10-01

    Inventors

    Cpc classification

    International classification

    Abstract

    Provided are a composition for detecting protein-protein interactions comprising fragments of secreted alkaline phosphatase (SEAP) and a method for detecting protein-protein interactions using the same. According to the composition or the method of the present invention, it is possible to simply detect the protein-protein interactions in the cells without environmental changes (e.g., cell destruction) in the cells. Furthermore, the composition or the method of the present invention can also be used for detection of materials that enhance or inhibit protein-protein interactions.

    Claims

    1. A composition for detecting protein-protein interactions comprising: a first construct comprising a polynucleotide encoding a first fusion protein comprising a bait protein and a secreted alkaline phosphatase (SEAP) first fragment protein; and a second construct comprising a polynucleotide encoding a second fusion protein comprising a prey protein and a SEAP second fragment protein.

    2. The composition of claim 1, wherein the SEAP is represented by the amino acid sequence of SEQ ID NO: 1.

    3. The composition of claim 1, wherein the SEAP first fragment protein and the SEAP second fragment protein are selected from the group consisting of fragments cleaved at amino acid positions 1 to 16, 52 to 68, 364 to 395, 396 to 426, or 473 to 489 from a N-terminal of the SEAP protein.

    4. The composition of claim 1, wherein the SEAP first fragment protein and the SEAP second fragment protein are selected from the group consisting of fragments cleaved at amino acid position 8, 60, 372, 379, 387, 404, 418, or 481 from a N-terminal of the SEAP protein.

    5. The composition of claim 1, wherein the SEAP first fragment protein and the SEAP second fragment protein are selected from the group consisting of fragments cleaved at amino acid positions 55 to 68 from a N-terminal of the SEAP protein.

    6. A method for detecting protein-protein interactions comprising: (a) introducing to cells a first construct comprising a polynucleotide encoding a first fusion protein comprising a bait protein and a secreted alkaline phosphatase (SEAP) first fragment protein; and a second construct comprising a polynucleotide encoding a second fusion protein comprising a prey protein and a SEAP second fragment protein; (b) expressing the fusion proteins and inducing protein-protein interactions; and (c) measuring SEAP activities before and after inducing the interactions.

    7. The method of claim 6, further comprising: (d) determining that the bait protein and the prey protein interact with each other when the SEAP activity after inducing the interaction measured in step (c) is increased compared to the SEAP activity before inducing the interaction.

    8. The method of claim 6, wherein the method analyzes the interactions between the bait protein and the prey protein in a time course.

    9. The method of claim 6, wherein the SEAP is represented by the amino acid sequence of SEQ ID NO: 1.

    10. The method of claim 6, wherein the SEAP first fragment protein and the SEAP second fragment protein are selected from the group consisting of fragments cleaved at amino acid positions 1 to 16, 52 to 68, 364 to 395, 396 to 426, or 473 to 489 from a N-terminal of the SEAP protein.

    11. The method of claim 6, wherein the SEAP first fragment protein and the SEAP second fragment protein are selected from the group consisting of fragments cleaved at amino acid positions 8, 60, 372, 379, 387, 404, 418, or 481 from a N-terminal of the SEAP protein.

    12. The method of claim 6, wherein the SEAP first fragment protein and the SEAP second fragment protein are selected from the group consisting of fragments cleaved at amino acid positions 55 to 68 from a N-terminal of the SEAP protein.

    13. A composition for screening a therapeutic agent comprising: a first construct comprising a polynucleotide encoding a first fusion protein comprising a bait protein and a secreted alkaline phosphatase (SEAP) first fragment protein; and a second construct comprising a polynucleotide encoding a second fusion protein comprising a prey protein and a SEAP second fragment protein.

    14. A composition for screening a promoter or inhibitor for protein-protein interactions comprising: a first construct comprising a polynucleotide encoding a first fusion protein comprising a bait protein and a secreted alkaline phosphatase (SEAP) first fragment protein; and a second construct comprising a polynucleotide encoding a second fusion protein comprising a prey protein and a SEAP second fragment protein.

    Description

    BRIEF DESCRIPTION OF DRAWINGS

    [0055] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

    [0056] FIG. 1 illustrates a secondary structure of a SEAP protein and cleavage positions according to the present invention. Yellow represents an -helix structure, red represents a -sheet structure, blue represents a turn structure, and green represents cleavage positions (starting positions of C-terminal fragments). (SEQ ID NO: 1) FIG. 2 is a schematic diagram illustrating vectors comprising a polynucleotide encoding a fusion protein in which a N-terminal fragment and a C-terminal fragment of the SEAP is linked to FKBP and FRB, respectively.

    [0057] FIG. 3 illustrates results of screening SEAP fragments capable of detecting protein-protein interactions.

    [0058] FIG. 4 illustrates results of measuring SEAP activities of pairs of SEAP fragments cleaved at each of amino acid positions 55 to 68 from a N-terminal of the SEAP protein.

    [0059] FIG. 5 illustrates results of measuring SEAP activities of pairs of a N-terminal fragment cleaved at amino acid position 59 from the N-terminal of the SEAP protein and each of C-terminal fragments cleaved at each of amino acid positions 55 to 65 from the N-terminal of the SEAP protein.

    [0060] FIG. 6 illustrates results of measuring SEAP activities of pairs of SEAP fragments cleaved at different positions.

    MODE FOR INVENTION

    [0061] Hereinafter, the present invention will be described in more detail with reference to Examples and Experimental Examples. However, these Examples and Experimental Examples are only illustrative of the present invention, and the scope of the present invention is not limited to these Examples and Experimental Examples.

    Example 1: Preparation of Vectors Expressing Fusion Protein Comprising SEAP Fragment

    Example 1-1: Determination of Cleavage Positions of SEAP

    [0062] Cleavage positions of SEAP consisting of the amino acid sequence of SEQ ID NO: I were selected from parts where a secondary structure was not confirmed in UniProtKB (ID P05187). In FIG. 1, the secondary structure and the cleavage positions of the SEAP were illustrated. Here, yellow represents an -helix structure, red represents a -sheet structure, blue represents a turn structure, and green represents cleavage positions (starting positions of C-terminal fragments).

    Example 1-2: Preparation of Vectors

    [0063] According to the cleavage position determined in Example 1-1, vectors (FIG. 2) encoding a fusion protein were prepared in which a N-terminal fragment of the SEAP was fused to a C-terminal of FKBP12 and a C-terminal fragment of the SEAP was fused to a N-terminal or C-terminal of FRB.

    [0064] The FKBP12 and the FRB were known to form a heterodimer mediated with rapamycin.

    [0065] The vectors used or prepared in Example 1-2 were illustrated in Table 1.

    [0066] The primers used in Example 1-2 were illustrated in Table 2.

    TABLE-US-00001 TABLE1 Vector Description Source pAH7 HA-FKBPexpressionvector Cosmogenetech (P.sub.hCMV-NheI-Ex4L-FC-XbaI-HA-FKBP-linker-BamHI-spacer- (Seoul,Korea) ApaI-pA.sub.bGH) P.sub.hCMV:Humancytomegalovirusimmediateearlypromoter Ex4L:exendin-4leadersequence (MKIILWLCVFGLFLATLFPISWQMPVESGLSSEDSASSES FAK(SEQIDNO:126)) FC:furincleavagesite(RIKR(SEQIDNO:127)) HA:hemagglutinintag(YPYDVPDYA(SEQIDNO:128)) Linker:KGSGSTSGSG(SEQIDNO:129); pAH8 FRB-FLAGexpressionvector Cosmogenetech (P.sub.hCMV-NheI-Ex4L-FC-XbaI-spacer-BamHI-linker-FRB-FLAG- (Seoul,Korea) ApaI-pA.sub.bGH) pAH9 FLAG-FRBexpressionvector Cosmogenetech (P.sub.hCMV-NheI-Ex4L-FC-XbaI-FLAG-FRB-linker-BamHI-spacer- (Seoul,Korea), ApaI-pA.sub.bGH) pSCA# FLAG-FRB-scSEAP#expressionvector Example1 (C-terminal (P.sub.hCMV-NheI-Ex4L-FC-XbaI-FLAG-FRB-linker-BamHI-scSEA fragment, P#-ApaI-pA.sub.bGH) #-502) scSEAP:splitC-terminalSEAPfragment pSCB# scSEAP#-FRB-FLAGexpressionvector Example1 (C-terminal (P.sub.hCMV-NheI-Ex4L-FC-XbaI-scSEAP#-BamHI-linker-FRB-FL fragment, AG-ApaI-pA.sub.bGH) #-502) pSNA# HA-FKBP-snSEAP#expressionvector Example1 (N-terminal (P.sub.hCMV-NheI-Ex4L-FC-XbaI-HA-FKBP-linker-BamHI-snSEAP fragment, #-ApaI-pA.sub.bGH). 1-#) snSEAP:splitN-terminalSEAPfragments pSEAPX SEAPexpressionvector Genscript (P.sub.hCMV-HindIII-SEAP-EcoRI-pA.sub.bGH) SEAPwassynthesizedbyremovingaBamHI/XbaIrestriction enzymecleavagesitewithoutachangeinaminoacidsinan encodingregion(BamHI:changedfromggatcctogaatcc, XbaI:changedfromtctagatotccaga). ThesynthesizedSEAPwassub-clonedtopUC57(pL1).The SEAPwascleavedfrompL1usingHindIII/EcoRIandinserted topcDNA3.1+usingacorrespondingregion.

    TABLE-US-00002 TABLE2 SEQ ID Primer Sequence(5.fwdarw.3) NO: oSCAR cagcgggtttaaacgggcccTCATGTCTGCTCGA 2 AGCGGCC oSCA9 tggaagtggaggatccCCGGACTTCTGGAACCGC 3 oSCA31 tggaagtggaggatccACAGCCGCCAAGAACCTC 4 oSCA45 tggaagtggaggatccGGGGTGTCTACGGTGACA 5 oSCA61 tggaagtggaggatccGACAAACTGGGGCCTGAG 6 oSCA70 tggaagtggaggatccGCCATGGACCGCTTCCCA 7 oSCA83 tggaagtggaggatccTACAATGTAGACAAACAT 8 GTGCC oSCA91 tggaagtggaggatccGACAGTGGAGCCACAGCC 9 oSCA103 tggaagtggaggatccGTCAAGGGCAACTTCCAG 10 oSCA115 tggaagtggaggatccGCCGCCCGCTTTAACCAG 11 oSCA128 tggaagtggaggatccGAGGTCATCTCCGTGATG 12 oSCA140 tggaagtggaggatccGGGAAGTCAGTGGGAGTG 13 oSCA152 tggaagtggaggatccCAGCACGCCTCGCCAGCC 14 oSCA162 tggaagtggaggatccCACACGGTGAACCGCAAC 15 oSCA169 tggaagtggaggatccTACTCGGACGCCGACGTG 16 oSCA176 tggaagtggaggatccGCCTCGGCCCGCCAGGAG 17 oSCA183 tggaagtggaggatccTGCCAGGACATCGCTACG 18 oSCA194 tggaagtggaggatccATGGACATTGACGTGATCC 19 oSCA210 tggaagtggaggatccATGGGAACCCCAGACCCT 20 oSCA219 tggaagtggaggatccGATGACTACAGCCAAGGT 21 oSCA230 tggaagtggaggatccGGGAAGAATCTGGTGCAG 22 oSCA242 tggaagtggaggatccCAGGGTGCCCGGTATGTG 23 oSCA249 tggaagtggaggatccAACCGCACTGAGCTCATG 24 oSCA260 tggaagtggaggatccCCGTCTGTGACCCATCTC 25 oSCA274 tggaagtggaggatccATGAAATACGAGATCCACCG 26 oSCA281 tggaagtggaggatccGACTCCACACTGGACCCCT 27 oSCA302 tggaagtggaggatccAACCCCCGCGGCTTCTTC 28 oSCA314 tggaagtggaggatccCGCATCGACCATGGTCAT 29 oSCA323 tggaagtggaggatccAGGGCTTACCGGGCACTG 30 oSCA346 tggaagtggaggatccAGCGAGGAGGACACGCTG 31 oSCA366 tggaagtggaggatccGGCTACCCCCTGCGAGGG 32 oSCA373 tggaagtggaggatccTCCATCTTCGGGCTGGCC 33 oSCA380 tggaagtggaggatccGGCAAGGCCCGGGACAGG 34 oSCA388 tggaagtggaggatccTACACGGTCTCCTATAC 35 oSCA405 tggaagtggaggatccGCCCGGCCGGATGTTACC 36 oSCA419 tggaagtggaggatccTATCGGCAGCAGTCAGCA 37 oSCA444 tggaagtggaggatccCCGCAGGCGCACCTGGTT 38 oSCA457 tggaagtggaggatccTTCATAGCGCACGTCATG 39 oSCA468 tggaagtggaggatccTGCCTGGAGCCCTACACC 40 oSCA474 tggaagtggaggatccTGCGACCTGGCGCCCCCC 41 oSCA482 tggaagtggaggatccACCACCGACGCCGCGCAC 42 oSCBR ctgaacctttggatccTGTCTGCTCGAAGCGGCC 43 oSCB9 catcaagcgctctagaCCGGACTTCTGGAACCGC 44 oSCB31 catcaagcgctctagaACAGCCGCCAAGAACCTC 45 oSCB45 catcaagcgctctagaGGGGTGTCTACGGTGACA 46 oSCB61 catcaagcgctctagaGACAAACTGGGGCCTGAG 47 oSCB70 catcaagcgctctagaGCCATGGACCGCTTCCCA 48 oSCB83 catcaagcgctctagaTACAATGTAGACAAACATGT 49 GCC oSCB91 catcaagcgctctagaGACAGTGGAGCCACAGCC 50 oSCB103 catcaagcgctctagaGTCAAGGGCAACTTCCAG 51 oSCB115 catcaagcgctctagaGCCGCCCGCTTTAACCAG 52 oSCB128 catcaagcgctctagaGAGGTCATCTCCGTGATG 53 oSCB140 catcaagcgctctagaGGGAAGTCAGTGGGAGTG 54 oSCB152 catcaagcgctctagaCAGCACGCCTCGCCAGCC 55 oSCB162 catcaagcgctctagaCACACGGTGAACCGCAAC 56 oSCB169 catcaagcgctctagaTACTCGGACGCCGACGTG 57 oSCB176 catcaagcgctctagaGCCTCGGCCCGCCAGGAG 58 oSCB183 catcaagcgctctagaTGCCAGGACATCGCTACG 59 oSCB194 catcaagcgctctagaATGGACATTGACGTGATCC 60 oSCB210 catcaagcgctctagaATGGGAACCCCAGACCCT 61 oSCB219 catcaagcgctctagaGATGACTACAGCCAAGGT 62 oSCB230 catcaagcgctctagaGGGAAGAATCTGGTGCAG 63 oSCB242 catcaagcgctctagaCAGGGTGCCCGGTATGTG 64 oSCB249 catcaagcgctctagaAACCGCACTGAGCTCATG 65 oSCB260 catcaagcgctctagaCCGTCTGTGACCCATCTC 66 oSCB274 catcaagcgctctagaATGAAATACGAGATCCACCG 67 oSCB281 catcaagcgctctagaGACTCCACACTGGACCCCT 68 oSCB302 catcaagcgctctagaAACCCCCGCGGCTTCTTC 69 oSCB314 catcaagcgctctagaCGCATCGACCATGGTCAT 70 oSCB323 catcaagcgctctagaAGGGCTTACCGGGCACTG 71 oSCB346 catcaagcgctctagaAGCGAGGAGGACACGCTG 72 oSCB366 catcaagcgctctagaGGCTACCCCCTGCGAGGG 73 oSCB373 catcaagcgctctagaTCCATCTTCGGGCTGGCC 74 oSCB380 catcaagcgctctagaGGCAAGGCCCGGGACAGG 75 oSCB388 catcaagcgctctagaTACACGGTCCTCCTATAC 76 oSCB405 catcaagcgctctagaGCCCGGCCGGATGTTACC 77 oSCB419 catcaagcgctctagaTATCGGCAGCAGTCAGCA 78 oSCB444 catcaagcgctctagaCCGCAGGCGCACCTGGTT 79 oSCB457 catcaagcgctctagaTTCATAGCGCACGTCATG 80 oSCB468 catcaagcgctctagaTGCCTGGAGCCCTACACC 81 oSCB474 catcaagcgctctagaTGCGACCTGGCGCCCCCC 82 oSCB482 catcaagcgctctagaACCACCGACGCCGCGCAC 83 oSNAF tggaagtggaggatccATCATCCCAGTTGAGGAG 84 oSNA8a gatccATCATCCCAGTTGAGGAGGAGAACTGAggg 85 cc oSNA8b cTCAGTTCTCCTCCTCAACTGGGATGATg 86 oSNA30 cagcgggtttaaacgggcccTCACTGTGCAGGCTGC 87 AGCTT oSNA44 cagcgggtttaaacgggcccTCACATCCCATCGCCC 88 AGGAA oSNA60 cagcgggtttaaacgggcccTCACTTCTTCTGCCCT 89 TTCAG oSNA69 cagcgggtttaaacgggcccTCACAGGGGTATCTCA 90 GGCCC oSNA82 cagcgggtttaaacgggcccTCATGTCTTGGACAGA 91 GCCAC oSNA90 cagcgggtttaaacgggcccTCATGGCACATGTTTG 92 TCTAC oSNA102 cagcgggtttaaacgggcccTCACCCGCACAGGTAG 93 GCCGT oSNA113 cagcgggtttaaacgggcccTCATGCACTCAAGCCA 94 ATGGT oSNA127 cagcgggtttaaacgggcccTCAGTTGCCGCGTGTC 95 GTGTT oSNA139 cagcgggtttaaacgggcccTCATGCTTTCTTGGCC 96 CGATT oSNA151 cagcgggtttaaacgggcccTCACACTCGTGTGGTG 97 GTTAC oSNA161 cagcgggtttaaacgggcccTCAGGCGTAGGTGCCG 98 GCTGG oSNA168 cagcgggtttaaacgggcccTCACCAGTTGCGGTTC 99 ACCGT oSNA175 cagcgggtttaaacgggcccTCAAGGCACGTCGGCG 100 TCCGA oSNA182 cagcgggtttaaacgggcccTCACCCCTCCTGGCGG 101 GCCGA oSNA193 cagcgggtttaaacgggcccTCAGTTGGAGATGAGC 102 TGCGT oSNA209 cagcgggtttaaacgggcccTCAGCGAAACATGTAC 103 TTTCG oSNA218 cagcgggtttaaacgggcccTCATGGGTACTCAGGG 104 TCTGG oSNA229 cagcgggtttaaacgggcccTCAGTCCAGCCTGGTC 105 CCACC oSNA241 cagcgggtttaaacgggcccTCAGCGCTTCGCCAGC 106 CATTC oSNA248 cagcgggtttaaacgggcccTCACCACACATACCGG 107 GCACC oSNA259 cagcgggtttaaacgggcccTCAGTCCAGGGAAGCC 108 TGCAT oSNA273 cagcgggtttaaacgggcccTCAGTCTCCAGGCTCA 109 AAGAG oSNA280 cagcgggtttaaacgggcccTCATCGGTGGATCTCG 110 TATTTC oSNA301 cagcgggtttaaacgggcccTCACCTGCTCAGCAGG 111 CGCAG oSNA313 cagcgggtttaaacgggcccTCAACCACCCTCCACG 112 AAGAG oSNA322 cagcgggtttaaacgggcccTCAGCTTTCATGATGA 113 CCATG oSNA345 cagcgggtttaaacgggcccTCAGGTGAGCTGGCCC 114 GCCCT oSNA365 cagcgggtttaaacgggcccTCATCCGAAGGAGAAG 115 ACGTG oSNA372 cagcgggtttaaacgggcccTCAGCTCCCTCGCAGG 116 GGGTA oSNA379 cagcgggtttaaacgggcccTCAAGGGGCCAGCCCG 117 AAGAT oSNA387 cagcgggtttaaacgggcccTCAGGCCTTCCTGTCC 118 CGGGC oSNA404 cagcgggtttaaacgggcccTCAGCCGTCCTTGAGC 119 ACATA oSNA418 cagcgggtttaaacgggcccTCACTCGGGGCTCCCG 120 CTCTC oSNA443 cagcgggtttaaacgggcccTCAGCCGCGCGCGAAC 121 ACCGC oSNA456 cagcgggtttaaacgggcccTCAGGTCTGCTCCTGC 122 ACGCC oSNA467 cagcgggtttaaacgggcccTCAGGCGGCGAAGGCC 123 ATGAC oSNA473 cagcgggtttaaacgggcccTCAGGCGGTGTAGGGC 124 TCCAG oSNA481 cagcgggtttaaacgggcccTCAGCCGGCGGGGGGC 125 GCCAG

    [0067] 1) Preparation of pSCA # (C-Terminal Fragment, #-502) Vector

    [0068] The vector is a vector expressing a FLAG-FRB-SEAP fragment (C-terminal).

    [0069] scSEAP (C-terminal fragment of SEAP) was amplified by PCR using a pSEAPX vector as a template and oSCA # and oSCAR as primers. The amplified PCR product and a pAH9 vector were cleaved with BamHI and ApaI restriction enzymes, and each cleaved product was ligated.

    [0070] 2) Preparation of pSCB # (C-Terminal Fragment, #-502) Vector

    [0071] The vector is a vector expressing a SEAP fragment (C-terminal)-FRB-FLAG.

    [0072] scSEAP (C-terminal fragment of SEAP) was amplified by PCR using a pSEAPX vector as a template and oSCB # and oSCBR as primers. The amplified PCR product and a pAH8 vector were cleaved with XbaI and BamHI restriction enzymes, and each cleaved product was ligated.

    [0073] 3) Preparation of pSNA # (N-Terminal Fragment, 1-#) Vector

    [0074] The vector is a vector expressing an HA-FKBP-SEAP fragment (N-terminal).

    [0075] snSEAP (N-terminal fragment of SEAP) was amplified by PCR using a pSEAPX vector as a template and oSNA # and oSNAF as primers. The amplified PCR product and a pAH7 vector were cleaved with BamHI and ApaI restriction enzymes, and each cleaved product was ligated.

    [0076] The pSNA8 vector was prepared by hybridizing oSNA8a and OSC8b primers and ligating the hybridized primers to the pAH7 cleaved with BamHI and ApaI.

    Example 2: Screening of SEAP Fragments Capable of Detecting Protein-Protein Interactions

    Example 2-1: Cell Culture

    [0077] HEK-293T (Human embryonic kidney cell, ATCC: CRL-11268) cells were cultured in DMEM (Dulbecco's modified Eagle's media, Gibco, Seoul, South Korea) treated with a 10% (v/v) FBS (HyClone) and 1% (v/v) penicillin/streptomycin solution (HyClone) and cultured at 37 C. in a humidified atmosphere containing 5% CO.sub.2.

    Example 2-2: Screening

    [0078] HEK-293T cells were seeded at 210.sup.4 cells per well in a 48-well plate and cultured until 24 hours before transformation.

    [0079] For the transformation, 0.15 L of PEI (PEI, <20,000 MW, Cat. No. 23966, Polysciences, Inc., Warrington, Pa., USA; stock solution: 4 mg/mL in ddH20, pH 7.2) was mixed with 0.2 pLg of DNA, and the mixture was vortexed for 5 seconds and then incubated for 20 minutes at 25 C. to prepare 40 L of DNA-PEI mixture per well. At this time, the DNA was prepared by mixing a vector (pSNA series) containing a SEAP N-terminal fragment and a vector (pSCA or pSCB series) containing a SEAP C-terminal fragment.

    [0080] After 24 hours of the transformation, the culture medium was replaced with DMEM with 100 nM rapamycin or DMEM without rapamycin.

    [0081] SEAP activity was measured after 24 hours. The SEAP activity was measured in a time course using a p-nitrophenylphosphate (pNpp)-based absorbance (405 nm) measuring method. 80 L of a culture medium supernatant, 100 L of a 2 SEAP buffer solution (21% diethanolamine, 20 mM L-homoarginine, and 1 mM MgCl.sub.2, pH 9.8), and 20 L of 120 mM pNpp were mixed and reacted with one another, and then absorbance at 405 nm was measured. The results were shown in FIG. 3.

    [0082] Referring to FIG. 3, it can be seen that when an interaction (binding) between FKBP12 and FRB in the cells was induced by treating with rapamycin, some fragments among various SEAP fragments fused to the FKBP12 or FRB were complemented with each other to exhibit SEAP activities.

    [0083] The SEAP fragment pairs binding to each other to exhibit the SEAP activities are pairs of fragments cleaved at amino acid positions 8, 60, 379, 404, or 481 from the N-terminal (FIG. 3). That is, the pairs of the SEAP fragments cleaved at amino acid positions 8, 60, 379, 404, or 481 from the N-terminal may be used to detect the protein-protein interactions.

    Example 3: Measurement of SEAP Activities of Pairs of SEAP Fragments Cleaved at Each of Amino Acid Positions 55 to 68 from N-Terminal

    [0084] As the results in the screening of Example 2, it can be seen that the pair of SEAP fragments cleaved at amino acid position 60 from the N-terminal is most excellent in SEAP activity (FIG. 3).

    [0085] Therefore, SEAP activities of pairs of SEAP fragments cleaved within 8 from amino acid position 60 were measured by an experiment in the same manner as Example 2, and the results thereof were illustrated in FIG. 4.

    [0086] Referring to FIG. 4, it can be seen that with the exception of the pair of SEAP fragments cleaved at amino acid position 55 from the N-terminal, all of the fragment pairs were complemented with each other to exhibit excellent SEAP activities. That is, the pairs of the SEAP fragments cleaved at each of amino acid positions 56 to 68 from the N-terminal may be used to detect the protein-protein interactions.

    Example 4: Measurement of SEAP Activities of Pairs of N-Terminal Fragment Cleaved at Amino Acid Position 59 from N-Terminal of SEAP Protein and C-Terminal Fragments Cleaved at Each of Amino Acid Positions 55 to 65 from N-Terminal of SEAP Protein

    [0087] SEAP activities of pairs of a N-terminal fragment pSNA59 cleaved at amino acid position 59 from a N-terminal of a SEAP protein and each of C-terminal fragments pSCA55 to pSCA65 cleaved at each of amino acid positions 55 to 65 from the N-terminal of the SEAP protein were measured by an experiment in the same manner as Example 2, and the results thereof were illustrated in FIG. 5.

    [0088] Referring to FIG. 5, it can be seen that the N-terminal fragment cleaved at amino acid position 59 from the N-terminal of the SEAP protein and each of the C-terminal fragments cleaved at each of amino acid positions 55 to 65 from the N-terminal of the SEAP protein were complemented with each other to exhibit SEAP activities. That is, the pairs of the N-terminal fragment cleaved at amino acid position 59 from the N-terminal of the SEAP protein and the each of C-terminal fragments cleaved at each of amino acid positions 55 to 65 from the N-terminal of the SEAP protein may be used to detect the protein-protein interactions.

    Example 5: Measurement of SEAP Activities of Pairs of SEAP Fragments Cleaved at Different Positions

    [0089] SEAP activities of pairs of SEAP fragments cleaved at different positions were measured by an experiment in the same manner as Example 2, and the results thereof were illustrated in FIG. 6.

    [0090] Referring to FIG. 6, it can be seen that a SEAP N-terminal fragment pSNA379 cleaved at amino acid position 379 from the N-terminal binds to a SEAP C-terminal fragment pSCA373 or pSCB373 cleaved at amino acid position 372 from the N-terminal to exhibit excellent SEAP activity.

    [0091] Further, it can be seen that a SEAP N-terminal fragment pSNA387 cleaved at amino acid position 387 from the N-terminal binds to a SEAP C-terminal fragment pSCA380 cleaved at amino acid position 379 from the N-terminal to exhibit excellent SEAP activity.

    [0092] Further, it can be seen that a SEAP N-terminal fragment pSNA404 cleaved at amino acid position 404 from the N-terminal binds to a SEAP C-terminal fragment pSCA388 or pSCB388 cleaved at amino acid position 387 from the N-terminal to exhibit excellent SEAP activity.

    [0093] Further, it can be seen that a SEAP N-terminal fragment pSNA418 cleaved at amino acid position 418 from the N-terminal binds to a SEAP C-terminal fragment pSCA405 or pSCB405 cleaved at amino acid position 404 from the N-terminal to exhibit excellent SEAP activity.

    [0094] That is, the pairs of fragments having different cleavage positions may be used to detect the protein-protein interactions.

    [0095] It will be appreciated by those skilled in the art that the present invention as described above may be implemented in other specific forms without departing from the technical spirit thereof or essential characteristics. Thus, it is to be appreciated that embodiments described above are intended to be illustrative in every sense, and not restrictive. The scope of the present invention is represented by the claims described below rather than the detailed description, and it is to be interpreted that the meaning and scope of the claims and all changes or modified forms derived from the equivalents thereof come within the scope of the present invention.