WO2017130610A1 - Protéine de fusion et procédé de détection d'antigène utilisant ladite protéine - Google Patents

Protéine de fusion et procédé de détection d'antigène utilisant ladite protéine Download PDF

Info

Publication number
WO2017130610A1
WO2017130610A1 PCT/JP2016/088061 JP2016088061W WO2017130610A1 WO 2017130610 A1 WO2017130610 A1 WO 2017130610A1 JP 2016088061 W JP2016088061 W JP 2016088061W WO 2017130610 A1 WO2017130610 A1 WO 2017130610A1
Authority
WO
WIPO (PCT)
Prior art keywords
mutant
fusion protein
region
lane
gus
Prior art date
Application number
PCT/JP2016/088061
Other languages
English (en)
Japanese (ja)
Inventor
上田　宏
金華 董
Original Assignee
国立大学法人東京工業大学
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 国立大学法人東京工業大学 filed Critical 国立大学法人東京工業大学
Priority to JP2017563741A priority Critical patent/JP6873483B2/ja
Publication of WO2017130610A1 publication Critical patent/WO2017130610A1/fr

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/34—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase

Definitions

  • the present invention relates to a fusion protein comprising an enzyme variant and an antibody variable region, and an antigen detection method and an antigen detection kit using the same.
  • Homogeneous immunoassay methods such as fluorescence polarization, EMIT, CEDIA, OS-FIA, and Quenchbody are known, and practical sensitivity comparable to that of conventional competitive methods can be obtained for small molecule detection.
  • fluorescence polarization EMIT, CEDIA, OS-FIA, and Quenchbody
  • the sensitivity is limited by the detection sensitivity of the fluorescent label, and there is a problem that signal amplification for improving sensitivity is difficult.
  • an enzyme that can improve sensitivity by signal amplification In many cases, the stability of the problem became a problem.
  • Non-patent Documents 1 and 2 using activity complementation between ⁇ -galactosidase deletion mutants have the advantage that the substrate is easy to use, but the enzyme stability, specific activity, and further activity The small amount of change was a big problem in practical use.
  • the homogeneous immunoassay has a merit that antigen can be detected easily and rapidly, but there are also problems with the stability and activity of the reagent.
  • the object of the present invention is to solve the problems of the conventional homogeneous immunoassay.
  • the present invention takes advantage of the property of ⁇ -glucuronidase, which exhibits enzyme activity only by forming a tetramer.
  • ⁇ -galactosidase is an enzyme that exhibits activity by forming a tetramer, but such properties are not used at all in the OS-ECIA method. Therefore, the present invention and the OS-ECIA method are based on completely different ideas. The present invention has been completed based on the above findings.
  • the present invention provides the following (1) to (8).
  • a fusion protein comprising a mutant of an enzyme activated by formation of a multimer, a linker peptide that binds to the mutant of the enzyme, and a V H region or a V L region of an antibody that binds to the linker peptide.
  • the fusion protein, wherein the mutant of the enzyme is a mutant into which a mutation that reduces the binding affinity between monomers is introduced.
  • the mutant of ⁇ -glucuronidase is a mutant in which the 516th methionine in the amino acid sequence of Escherichia coli ⁇ -glucuronidase is replaced with lysine and the 517th tyrosine is replaced with glutamic acid.
  • a method for detecting an antigen in a sample comprises a V L region of a fusion protein and antibody according to any one of including the V H region of an antibody (1) to (5)
  • a method for detecting an antigen comprising the steps of contacting the fusion protein according to any one of (1) to (5) and detecting the formation of multimers by a change in enzyme activity.
  • the antigen detection method of the present invention can detect an antigen simply and rapidly, and is excellent in terms of detection sensitivity.
  • lane M 100 bp DNA ladder
  • lane 1 GUS gene
  • lane 2 mutated GUS gene fragment (5 'side)
  • lane 3 mutated GUS gene fragment (3' side)
  • lane 4 mutated GUS gene.
  • lane M DNA marker, lane 1: GUS mutant gene (after restriction enzyme treatment), lane 2: GUS mutant gene (before restriction enzyme treatment), lane 3: V H (NP) vector (after restriction enzyme treatment), lane 4: V H (NP) vector (before restriction enzyme treatment), lane 5: V L (NP) vector (after restriction enzyme treatment), lane 6: V L (NP) vector (before restriction enzyme treatment).
  • B lane M: DNA marker, lane 1-4: amplified (G 4 S) 3 gene.
  • Lane 1 pET32-V H (NP) -GUS vector before restriction enzyme treatment
  • lane 2 pET32-V H (NP) -GUS vector treated with NotI
  • lane 3 pET32 treated with HindIII -V H (NP) -GUS vector.
  • B Electrophoresis photograph.
  • lane M protein molecular weight marker
  • lane 1 whole cell protein before inducing expression
  • lane 2 whole cell protein after inducing expression.
  • the arrow indicates the target protein.
  • A Purification by TALON-immobilized metal affinity chromatography, lane M: protein molecular weight marker, lane 1-5: V H (NP)-(G 4 S) 3 -GUS mutant, lane 6-10: V L ( NP)-(G 4 S) 3 -GUS mutant, lane 1,6: insoluble fraction, lane 2,7: soluble fraction, lane 3,8: flow-through, lane 4, 5, 9, 10: elution Fraction.
  • lane M protein molecular weight marker
  • lane 1 total intracellular protein before induction of expression
  • lane 2 total intracellular protein after expression induction
  • lane 3 insoluble fraction
  • lane 4 soluble fraction
  • lane 5 flow Through
  • lane 6 eluted protein.
  • the fusion protein of the present invention comprises a mutant of an enzyme activated by formation of a multimer, a linker peptide that binds to the mutant of the enzyme, and a VH region of an antibody that binds to the linker peptide.
  • it is a fusion protein containing a VL region, wherein the mutant of the enzyme is a mutant into which a mutation that reduces the binding affinity between monomers is introduced.
  • “An enzyme activated by the formation of a multimer” means an enzyme that shows an activity or an improvement in activity only when several monomers are bonded.
  • the number of monomers constituting the multimer is not particularly limited, and may be an enzyme activated by any of dimer, trimer, tetramer, pentamer, hexamer, and the like.
  • the enzyme activated by the formation of multimers is preferably an enzyme whose activity can be easily detected or measured. For example, an enzyme that can detect or measure a product or substrate by absorbance, fluorescence intensity, or luminescence intensity.
  • enzymes activated by the formation of multimers include those generally used as reporter enzymes, such as ⁇ -glucuronidase (activated by tetramer), ⁇ -galactosidase (activated by tetramer). Alkaline phosphatase (activated by dimer), malate dehydrogenase (activated by dimer), and the like.
  • Examples of the mutation that reduces the binding affinity between monomers include a mutation introduced at a binding site between monomers. Many of the enzymes that are activated by the formation of multimers have been clarified in their amino acid sequences and binding sites between monomers. I understand how affinity can be reduced. The degree of the affinity decrease may be such that it is difficult to form a multimer due to a decrease in the binding affinity between the monomers, thereby recognizing the difference in activity from the wild-type enzyme. Mutants that have introduced mutations that reduce the binding affinity between monomers include those that have reduced the affinity of all bonds between monomers, as well as the affinity of some bonds between monomers. Also included are those that only declined.
  • a mutant that forms a dimer but does not easily form a tetramer such as a mutant of ⁇ -glucuronidase described later, is introduced with a mutation that reduces the binding affinity between the monomers. Included in the mutant.
  • mutations that reduce the binding affinity between monomers include mutants in which the 516th methionine and the 517th tyrosine in the amino acid sequence of Escherichia coli ⁇ -glucuronidase are substituted with other amino acids.
  • the other amino acid substituted with the 516th methionine includes lysine
  • the other amino acid substituted with the 517th tyrosine includes glutamic acid.
  • the 516th methionine and the 517th tyrosine indicate the position in the amino acid sequence of ⁇ -glucuronidase derived from Escherichia coli, the methionine and tyrosine are not present in the aforementioned positions in the amino acid sequence of ⁇ -glucuronidase derived from other organisms. There is also. In such a case, the amino acid sequence is aligned with the amino acid sequence of ⁇ -glucuronidase derived from E. coli, and methionine and tyrosine corresponding to 516th methionine and 517th tyrosine are substituted with other amino acids. Like that.
  • the amino acid sequence of wild-type ⁇ -glucuronidase derived from E. coli is shown in SEQ ID NO: 1, and the amino acid sequence of ⁇ -glucuronidase into which the above mutation has been introduced is shown in SEQ ID NO: 2.
  • Any linker may be used as long as the enzyme, V H region, and VL region can function normally. If the distance between the enzyme and the V H region or VL region is not sufficient, the enzyme may not exhibit activity, so the linker needs to have a certain length.
  • the length of the linker varies depending on the type of enzyme and antibody used, but is usually 10 to 60 mm, preferably 30 to 40 mm.
  • the number of amino acids in the linker is not limited as long as it is the above-mentioned length, but is usually 5 to 50, preferably 15 to 20.
  • the amino acid sequence of the linker may be the same as the amino acid sequence of a general linker used in the production of a fusion protein.
  • Gly-Gly-Gly-Gly-Gly-Ser (G 4 S) repetitive sequence (repetition number is usually 2-5), Glu-Ala-Ala-Ala-Lys (EAAAK) repetitive sequence, Asp- Asp-Ala-Lys-Lys (DDAKK) repetitive sequence and the like can be mentioned.
  • the antibody V H region or V L region can be selected from any antibody V H region or V L region according to the antigen to be detected, and is limited to a specific antibody V H region or V L region. Not. Specifically, the V H region or VL region of an antibody that specifically binds to the antigen to be detected, which will be described later, can be used.
  • the fusion protein of the present invention may consist of only three of the above-mentioned enzyme mutant activated by the formation of a multimer, linker peptide, VH region or VL region, but other peptides and proteins, etc. May be included.
  • peptides include tag sequences for purification such as His-Tag, tag sequences that solubilize expressed proteins such as thioredoxin and amyloid precursor protein-derived solubilized tag sequences, and the like.
  • an enzyme mutant activated by multimer formation a linker peptide, a V H region or a VL region are arranged in this order.
  • the V H region or VL region may be the C terminus at the N terminus, and conversely, the mutant of the enzyme activated by formation of a multimer may be the C terminus and the V H region or VL region may be the N terminus.
  • One mutant of the enzyme that is activated by the formation of a multimer may be included in the fusion protein, but may be included in two or more. When two variants of the enzyme are included, they are arranged adjacent to each other via a linker. The linker used at this time may be the same as the linker disposed between the mutant of the enzyme and the VH region or VL region.
  • nucleic acid mainly refers to deoxyribonucleic acid, but also includes ribonucleic acid and modifications of these nucleic acids.
  • the antigen detection method of the present invention is a method for detecting an antigen in a sample, the sample comprising the fusion protein of the present invention containing the antibody V H region and the antibody V L region. Including the step of contacting with the fusion protein of the present invention, and the step of detecting the formation of multimers.
  • This method is based on the principle of the open sandwich method developed by the present inventors (H. Ueda et al., Nat. Biotechnol. 14 (12), 1714-1718 (1996)), that is, V H region and V L It takes advantage of the principle that domain interactions are enhanced by the presence of antigen.
  • the conventional antigen detection method using the open sandwich method for example, Non-Patent Documents 1 and 2
  • an N-terminal deletion mutant and a C-terminal deletion mutant are used.
  • multimer formation is performed.
  • Antigen detection is performed using a mutant of the enzyme activated by.
  • ⁇ -glucuronidase becomes active by forming a tetramer.
  • the above-mentioned mutant of ⁇ -glucuronidase forms a dimer because a mutation that reduces the binding affinity between some monomers is introduced, but does not form a tetramer under normal conditions. . If the sample does not exist antigen, since the interaction of V H and V L domains is a weak remains almost tetramer dimers in intact variants of ⁇ -glucuronidase which connects the V H region and the V L region It will not be.
  • the interaction between the V H region and the VL region is enhanced, and this interaction also causes a ⁇ -glucuronidase mutant dimer linked to the V H region and the VL region. It binds to form a tetramer and becomes active. Therefore, by measuring ⁇ -glucuronidase activity, it can be determined whether or not the antigen is present in the sample.
  • any antigen can be detected, but the method of the present invention is suitable for detecting low molecular weight compounds (for example, compounds having a molecular weight of 1000 or less). Is preferred. Further, since the method of the present invention can be used for disease diagnosis, food toxicity test, environmental analysis, and the like, it is preferable to use substances related to these as detection targets.
  • neonicotinoid pesticides such as imidacloprid
  • environmental pollutants such as polychlorinated biphenyls and bisphenol A
  • toxic substances such as mycotoxins, osteocalcin (effective for diagnosis of osteoporosis), corticoids, estradiol, aldosterone, lysozyme (chicken) And biological substances such as egg white lysozyme) and drugs such as digoxin.
  • the sample may be any sample that may contain the antigen to be detected.
  • human samples blood, saliva, urine, etc.
  • contaminated water examples include soil, food, and food ingredients.
  • the method for contacting the sample and the fusion protein is not particularly limited, it is usually performed by allowing the sample and the fusion protein to coexist in the solution. Moreover, you may coexist with the cell which expresses not a fusion protein itself but a fusion protein. Conditions such as temperature, time, pH of the solution, and the amount of the fusion protein to be used in this contact step may be those generally used for the enzyme contained in the fusion protein.
  • the temperature in this contact step is preferably about 20 to 37 ° C.
  • the contact time is preferably about 10 to 60 minutes
  • the pH of the solution is 6.8 to About 7.5 is preferable
  • the concentration of the fusion protein in the solution is preferably about 10 to 100 nM.
  • the formation of multimers can be detected by a change (increase or expression) in the activity of the enzyme mutant contained in the fusion protein.
  • the activity of the enzyme variant contained in the fusion protein can be measured by an activity measurement method generally used for the enzyme.
  • the activity can be measured by adding a chromogenic substrate or a fluorescent substrate and quantifying a substance produced from the substrate.
  • chromogenic substrates for ⁇ -glucuronidase include X-Gluc, 4-nitrophenyl ⁇ -glucopyranoside, 4-nitrophenyl ⁇ -D-glucuronide
  • fluorescent substrates include 4-methylumbelliferyl- ⁇ -D- Examples include glucuronide.
  • Quantification of substances produced from these substrates can be performed by measuring absorbance, fluorescence intensity, etc. at a specific wavelength.
  • the product can be quantified by measuring the absorbance near 405 nm, and if the substrate is 4-methylumbellylphenyl- ⁇ -D-glucuronide, 340 The product can be quantified by exciting with nm fluorescence and measuring the fluorescence intensity around 480 nm.
  • the antigen detection kit of the present invention comprises the fusion protein of the present invention containing the V H region of an antibody and the fusion protein of the present invention containing the VL region of an antibody. This kit can detect an antigen in a sample according to the principle of antigen detection described above.
  • This kit may contain other than the fusion protein of the present invention containing the V H region of the antibody and the fusion protein of the present invention containing the VL region of the antibody.
  • a substrate since a substrate is required for measuring enzyme activity, it may be included. Further, it may contain a reagent or equipment for quantifying a substance produced from the substrate, or a fusion protein or a substance for stabilizing the substrate.
  • FIG. 1B The primer sequence (5′-3 ′) used is shown in FIG. 1B. Specifically, first, the 5 ′ side of the GUS gene was amplified by polymerase chain reaction (PCR) using primer a (SEQ ID NO: 3) and primer d (SEQ ID NO: 6). Further, the 3 ′ side of the GUS gene was amplified by PCR using primer c (SEQ ID NO: 5) and primer b (SEQ ID NO: 4). Primers c and d contain the codon sequence of the amino acid mutation to be introduced.
  • PCR polymerase chain reaction
  • GUSm GUS mutant gene
  • KOD-Plus-Neo polymerase was used to first denature the DNA by incubating at 94 ° C for 2 minutes, and then 30 cycles of 94 ° C for 30 seconds, 55 ° C for 30 seconds, and 68 ° C for 1 minute were performed. Gel electrophoresis was performed to confirm the amplified DNA sample. The result is shown in FIG.
  • Lane 1 is amplified using primers a and b using the GUS gene before mutagenesis as a template
  • Lane 2 is the 5 'fragment of the mutagenized GUS gene
  • Lane 3 is the 3' fragment of the mutagenized GUS gene
  • Lane 4 is a mutant GUS gene amplification product.
  • restriction sites NotI and XhoI were cleaved at the 5 ′ and 3 ′ sides of the GUS gene.
  • Example 2 Construction of GUS mutant expression vector
  • the GUS gene and mutant gene prepared in Example 1 were cloned into an expression vector.
  • the amplified GUS gene DNA was digested with restriction enzymes NotI and XhoI.
  • E. coli expression vectors pET-VH (NP) -Rluc and pET-VL (NP)-(G3S) 3 encoding high affinity anti-NP (4-hydroxy-3-nitrophenyl acetic acid: molecular weight 198) antibody variable region -EYFP (Non-patent Document 1) was treated with NotI and XhoI, respectively, and confirmed by agarose gel electrophoresis. The results are shown in FIG.
  • Lane M is a molecular weight marker, and lanes 1 and 2 show GUS mutant genes after and before restriction enzyme treatment.
  • Lanes 3 and 4 are pET-VH (NP) -Rluc vectors after and before restriction enzyme treatment, and lanes 5 and 6 are pET32-VL (NP)-(G4S) 3-EYFP vectors after and before restriction enzyme treatment. .
  • NP pET32-VH
  • NP pET32-VL
  • G4S 3-GUSm
  • KOD-Plus-Neo polymerase was used to incubate at 94 ° C. for 2 minutes to denature the DNA. Thereafter, a reaction of 94 ° C. for 30 seconds, 55 ° C. for 30 seconds, and 68 ° C. for 1 minute was performed for 30 cycles, and agarose gel electrophoresis was performed to confirm the amplified DNA sample. The result is shown in FIG. 4B.
  • the amplified GS linker gene fragment was treated with HindIII / NotI and ligated with a vector (FIG. 4C) treated with the same enzyme set with T4 ligase to construct pET32-VH (NP)-(G4S) 3-GUSm. .
  • Example 4 Expression of V H (NP)-(G 4 S) 3 -GUSm and V L (NP)-(G 4 S) 3 -GUSm pET32-VH (NP)-(G4S) 3-GUSm and pET32-VL (NP)-(G4S) 3-GUSm was used to transform E. coli SHuffle T7 lysY. After culturing the plasmid-carrying E. coli in 500 ml of LBA medium (10 g / L tryptone, 5 g / L yeast, 5 g / L NaCl, 100 ⁇ g / mL ampicillin) at 30 ° C.
  • LBA medium 10 g / L tryptone, 5 g / L yeast, 5 g / L NaCl, 100 ⁇ g / mL ampicillin
  • Lane M is a protein molecular weight marker
  • lane 1 is the total intracellular protein before induction of expression
  • lane 2 is the total intracellular protein after induction of expression.
  • the arrow indicates the target protein.
  • Example 5 Purification of V H (NP)-(G 4 S) 3 -GUSm and V L (NP)-(G 4 S) 3 -GUSm 40 ml Extraction buffer (50 mM sodium phosphate, 300 mM Escherichia coli suspended in sodium chloride, pH 7.0) was crushed with a sonicator, centrifuged at 1000 g for 20 minutes, and the supernatant was collected and purified by immobilized metal affinity chromatography. Specifically, an appropriate amount of TALON (Takara Bio, Clontech) agarose gel was added to the supernatant and stirred for 2 hours.
  • TALON Tikara Bio, Clontech
  • Lane M shows molecular weight markers
  • lanes 1-5 show V H (NP)-(G 4 S) 3 -GUS mutants.
  • Lane 1 is the insoluble fraction
  • lane 2 is the soluble fraction
  • lane 3 is the flow-through
  • lanes 4 and 5 are the eluted protein.
  • Lanes 6-10 show V L (NP)-(G 4 S) 3 -GUS mutants.
  • Lane 6 shows the insoluble fraction
  • lane 7 is the soluble fraction
  • lane 8 is the flow-through
  • lanes 9 and 10 are the eluted protein.
  • V H (NP)-(G 4 S) 3 -GUSm gave a band of almost single molecular weight ( ⁇ 100 kd)
  • V L (NP)-(G 4 S) 3 -GUSm In the case of Nd, a band of contaminants that seemed to contain N-terminal thioredoxin tag (14 kd), V L (12 kd) and GUS degradation products was observed in the vicinity of 35 kd.
  • V L (NP)-(G 4 S) 3 -GUSm was subsequently purified using an anion exchange resin, and finally a protein consisting of a single band was obtained.
  • FIG. 6B Lane 11 is a concentrated TALON purified elution fraction, and lanes 12-15 show a fraction eluted with NaCl.
  • Example 7 Antigen concentration and GUS activity (fluorescence) response A solution containing 50 nM each of V H (NP)-(G 4 S) 3 -GUSm and V L (NP)-(G 4 S) 3 -GUSm To the final concentration of 5, 10, 50, 100, 500, 1000, 5000, 10000 nM and add NP or NIP (5-iodo-NP, which binds to the antibody about 10 times stronger than NP), and 25 Incubated for 10 minutes at ° C.
  • NP or NIP 5-iodo-NP, which binds to the antibody about 10 times stronger than NP
  • the fluorescent substrate 4-methylumbellylphenyl- ⁇ -D-glucuronide (MUG, Wako Pure Chemical Industries, Ltd.) was added and incubated in a black half-well microplate at 25 ° C for 15 minutes, then excited at 340 nm and excited at 480 nm. The fluorescence intensity of was measured. Then, NP and NIP calibration curves based on the fluorescence intensity at each concentration were prepared. As shown in FIG. 8, as the NP and NIP concentrations increased, the fluorescence intensity gradually increased. When 10 ⁇ M NIP was added, the fluorescence intensity increased 5 times or more compared to when no antigen was added.
  • MUG 4-methylumbellylphenyl- ⁇ -D-glucuronide
  • Example 8 Construction of GUS mutant expression vector pET32-VH (NP)-(G4S) 3-GUSm and pET32-VL (NP)-(G4S) 3-GUSm constructed in Examples 2 and 3 (FIG. 9A) In order to insert the VH (BGP) and VL (BGP) genes into), the following operation was performed.
  • primers a and b VH (KTM) NcoBack: 5'-ATATGCCATGGATCAAGTAAAGCTGCAGCAGTC-3 '(SEQ ID NO: 9), VH_HindFor: 5'-CCCAAGCTTGCTCGAGAGACGGTGACCGT-3' (SEQ ID NO: 10)) and primers c, d (Vk (KTM) NcoSalBack: 5'-CATGCCATGGGGTCGACGGACATTGAGCTCACCCAG-3 '(SEQ ID NO: 11), Vk_HindFor: 5'-CCCAAGCTTCCGTTTTATTTCCAGCTT-3' (SEQ ID NO: 12)) (BGP) and VL (BGP) genes were amplified.
  • VLBGPback_to_pRSETSA (Bam): 5'- GGAGGAGGTGGGATCCATGGGGTCGACGGACATTG-3 '(SEQ ID NO: 13)) and primer f (GUSmutXhoFor_to_pRSETSA (Hd): 5'- CAGCCGGATCAAGCTCTCGAGTAGTCATTGTTTGC-3' (Example 14)
  • VL (BGP)-(G4S) 3-GUSm was amplified using pET32-VL (BGP)-(G4S) 3-GUSm constructed in step 1 as a template.
  • KOD-Plus-Neo polymerase was used to incubate at 94 ° C.
  • FIG. 10C Amplified VL (BGP)-(G4S) 3-GUSm was treated with BamHI and HindIII, ligated with pRsetSA plasmid (Fig. 10B, Dr. Tetsuya Kitaguchi, Waseda University) treated with the same enzyme set, and pRsetSA -VL (BGP)-(G4S) 3-GUSm was constructed.
  • Lane M is a protein molecular weight marker
  • lane 1 is the total intracellular protein before induction of expression
  • lane 2 is the total intracellular protein after induction of expression.
  • Escherichia coli suspended in 40 ml of Extraction buffer 50 mM sodium phosphate, 300 mM sodium chloride, pH 7.0
  • Extraction buffer 50 mM sodium phosphate, 300 mM sodium chloride, pH 7.0
  • TALON Tekara Bio, Clontech
  • the column was transferred to a column and washed three times with 10 ml of extraction buffer, and the protein bound to the gel was eluted using 2.5 ml of extraction buffer containing 150 mM imidazole.
  • Lane 3 is the insoluble fraction
  • lane 4 is the soluble fraction
  • lane 5 is the flow-through
  • lane 6 is the eluted protein.
  • V H (BGP)-(G 4 S) 3 -GUSm and V L (BGP)-(G 4 S) 3 -GUSm gave bands of almost single molecular weight ( ⁇ 100 kd). The arrow indicates the target protein.
  • Example 11 Change in GUS activity (absorbance) with and without antigen over time V H (BGP)-(G 4 S) 3 -GUSm and V L (BGP)-(G 4 S) 3 -GUSm
  • the buffer was exchanged with an acid buffer (pH 7.4) to prepare a final concentration of 0.1 ⁇ M.
  • Antigen BGP-C7 (7-residue peptide at the C-terminal of osteocalcin) was added to a final concentration of 10 ⁇ M and incubated at 25 ° C.
  • Example 12 Antigen Concentration and GUS Activity Response
  • V H (BGP)-(G 4 S) 3 -GUSm and V L (BGP)-(G 4 S) 3 -GUSm the final concentration is 0, BGP-C7 was added to 1, 10, 10 2 , 10 3 , 10 4 , 10 5 , 10 6 nM and incubated at 25 ° C. for 10 minutes.
  • the fluorescent substrate 4-methylumbellylphenyl- ⁇ -D-glucuronide (MUG, Wako Pure Chemical Industries) was added, incubated in a black half-well microplate for 15 minutes at 25 ° C., then excited at 340 nm and 480 nm The fluorescence intensity at was measured.
  • a calibration curve for BGP-C7 based on the fluorescence intensity at each concentration was prepared.
  • the absorbance at 655 nm in each well was reduced as a background, and a calibration curve for BGP-C7 based on the absorbance at each concentration was prepared.
  • the fluorescence intensity and absorbance gradually increased as the BGP-C7 concentration increased.
  • Example 13 Comparison of activity between GUS mutant and wild-type GUS Fusion protein (V H (NP)-(G 4 S) 3 -GUSm and V L (NP) containing the GUS mutant prepared in Example 5 -(G 4 S) 3 -GUSm) and the enzyme activity of a fusion protein containing wild-type GUS (a fusion protein in which the GUS mutant in the fusion protein containing the GUS mutant prepared in Example 5 is replaced with wild-type GUS) It measured by the following method.
  • Both fusion proteins were mixed with 1 ⁇ M NP in PBST buffer, and 1 mg / mL enzyme substrate (4-nitrophenyl ⁇ -D-glucuronide) was added thereto. After incubating for 10 minutes until the reaction rate became stable, the absorbance at 405 nm was measured every 15 seconds for 15 minutes using a spectrophotometer (Beckmann DU530).
  • FIG. 14 shows the time course of absorbance when a fusion protein containing a GUS mutant is used
  • FIG. 15 shows the time course of absorbance when a fusion protein containing wild-type GUS is used.
  • the absorbance change when using a fusion protein containing a GUS mutant is 0.0096 (min -1 )
  • the absorbance change when using a fusion protein containing wild-type GUS is 0.0188 (min -1 )Met. From these values, the molar extinction coefficient of the reaction product p-nitrophenol (18.3 mM ⁇ 1 cm ⁇ 1 ), and the protein weight, the specific activities of both fusion proteins were calculated according to the Lambert Beer method.
  • the specific activity of the fusion protein containing wild-type GUS was 0.55 ⁇ molmin -1 mg -1 , whereas the specific activity of the fusion protein containing the GUS mutant was 0.043 ⁇ molmin -1 mg -1 The activity of about 1/13 of the fusion protein was maintained.
  • Non-Patent Document 1 T. Yokozeki, H. Ueda, R. Arai, W. Mahoney and T. Nagamune. Anal. Chem. 74, 2500-2504 (2002)
  • the activity of the mutant is wild type. It is described that the activity is reduced to 6.0 ⁇ 10 ⁇ 3 times (0.6%) at the maximum
  • Mohler WA, Blau HM (1996) Proc. Natl. Acad. Sci. USA 93: 12423- 12427 describes that the mutant is 25-200 times weaker than the wild type when compared to intracellular activity.
  • the above-mentioned activity exhibited by the fusion protein containing the GUS mutant is very high.
  • the present invention is useful for disease diagnosis, food inspection, environmental analysis, etc., it can be used in industrial fields related to these.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Engineering & Computer Science (AREA)
  • Wood Science & Technology (AREA)
  • Zoology (AREA)
  • Biochemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Health & Medical Sciences (AREA)
  • Molecular Biology (AREA)
  • Biotechnology (AREA)
  • General Engineering & Computer Science (AREA)
  • Biophysics (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Medicinal Chemistry (AREA)
  • Microbiology (AREA)
  • Biomedical Technology (AREA)
  • Immunology (AREA)
  • Physics & Mathematics (AREA)
  • Analytical Chemistry (AREA)
  • Plant Pathology (AREA)
  • Enzymes And Modification Thereof (AREA)
  • Peptides Or Proteins (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

L'invention vise à établir un moyen de détection d'antigène apte à détecter simplement et rapidement un antigène et présentant une excellente sensibilité de détection, etc. La protéine de fusion comprend un variant enzymatique qui est activé par la formation de multimères, un peptide lieur lié au variant enzymatique, et le domaine VH ou le domaine VL d'un anticorps qui se relie au peptide lieur, et est caractérisée en ce que le variant enzymatique présente une mutation introduite qui réduit l'affinité de liaison entre les monomères.
PCT/JP2016/088061 2016-01-25 2016-12-21 Protéine de fusion et procédé de détection d'antigène utilisant ladite protéine WO2017130610A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
JP2017563741A JP6873483B2 (ja) 2016-01-25 2016-12-21 融合タンパク質及びそれを用いた抗原の検出方法

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
JP2016-011475 2016-01-25
JP2016011475 2016-01-25

Publications (1)

Publication Number Publication Date
WO2017130610A1 true WO2017130610A1 (fr) 2017-08-03

Family

ID=59398096

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/JP2016/088061 WO2017130610A1 (fr) 2016-01-25 2016-12-21 Protéine de fusion et procédé de détection d'antigène utilisant ladite protéine

Country Status (2)

Country Link
JP (1) JP6873483B2 (fr)
WO (1) WO2017130610A1 (fr)

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2020068746A (ja) * 2018-11-02 2020-05-07 国立大学法人東京工業大学 バイオセンサー
WO2020162203A1 (fr) * 2019-02-08 2020-08-13 国立大学法人東京工業大学 Mutant enzymatique pour méthode d'immunoessai homogène
JP2020130167A (ja) * 2019-02-15 2020-08-31 国立大学法人宇都宮大学 タンパク質の発現方法およびタンパク質発現用ベクター

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH1078436A (ja) * 1996-07-31 1998-03-24 Boehringer Mannheim Corp 抗原濃度測定方法

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH1078436A (ja) * 1996-07-31 1998-03-24 Boehringer Mannheim Corp 抗原濃度測定方法

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
GEDDIE, M. L. ET AL.: "Antibody- induced oligomerization and activation of an engineered reporter enzyme", JOURNAL OF MOLECULAR BIOLOGY, vol. 369, no. 4, 2007, pages 1052 - 1059, XP022083580 *
HIROSHI UEDA ET AL.: "Jinko Allosteric Koso no Soshutsu ni yoru Kakushu Kokando Homogeneous Men'eki Sokuteiho no Kaihatsu", ABSTRACTS OF THE 68TH ANNUAL MEETING OF THE SOCIETY FOR BIOTECHNOLOGY, 25 August 2016 (2016-08-25), Japan, pages 160 *
UEDA, H. ET AL.: "An optimized homogeneous noncompetitive immunoassay based on the antigen-driven enzymatic complementation", JOURNAL OF IMMUNOLOGICAL METHODS, vol. 279, no. 1-2, 2003, pages 209 - 218, XP004455321 *
YOKOZEKI, T. ET AL.: "A homogeneous noncompetitive immunoassay for the detection of small haptens", ANALYTICAL CHEMISTRY, vol. 74, no. 11, 2002, pages 2500 - 2504, XP008156580 *

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2020068746A (ja) * 2018-11-02 2020-05-07 国立大学法人東京工業大学 バイオセンサー
WO2020090974A1 (fr) * 2018-11-02 2020-05-07 国立大学法人東京工業大学 Biocapteur
JP7169582B2 (ja) 2018-11-02 2022-11-11 国立大学法人東京工業大学 バイオセンサー
WO2020162203A1 (fr) * 2019-02-08 2020-08-13 国立大学法人東京工業大学 Mutant enzymatique pour méthode d'immunoessai homogène
JP2020130167A (ja) * 2019-02-15 2020-08-31 国立大学法人宇都宮大学 タンパク質の発現方法およびタンパク質発現用ベクター

Also Published As

Publication number Publication date
JPWO2017130610A1 (ja) 2018-11-15
JP6873483B2 (ja) 2021-05-19

Similar Documents

Publication Publication Date Title
US10844099B2 (en) Streptavidin muteins and methods of using them
Nallamsetty et al. Gateway vectors for the production of combinatorially‐tagged His6‐MBP fusion proteins in the cytoplasm and periplasm of Escherichia coli
US7960312B2 (en) Method and agent for immobilizing protein via protein bound to silicon oxide-containing substance
Iwanczyk et al. Role of the PDZ domains in Escherichia coli DegP protein
WO2017130610A1 (fr) Protéine de fusion et procédé de détection d'antigène utilisant ladite protéine
Schwarz et al. Using an E. coli Type 1 secretion system to secrete the mammalian, intracellular protein IFABP in its active form
KR20220144822A (ko) 재조합 칼프로텍틴
Hwang et al. Association of the Rad9–Rad1–Hus1 checkpoint clamp with MYH DNA glycosylase and DNA
Wijekoon et al. Evaluation of employing poly-lysine tags versus poly-histidine tags for purification and characterization of recombinant copper-binding proteins
Zhang et al. Novel signal-on immunosensors for rapid and sensitive detection of Microcystin-LR
Lee et al. The Dac-tag, an affinity tag based on penicillin-binding protein 5
EP2756081B1 (fr) Réactifs de dosage thermostables
WO2020090974A1 (fr) Biocapteur
Choi et al. Non‐allosteric enzyme switches possess larger effector‐induced changes in thermodynamic stability than their non‐switch analogs
Raghava et al. Refolding and simultaneous purification by three‐phase partitioning of recombinant proteins from inclusion bodies
JP5137266B2 (ja) 抗原濃度測定法
US8586315B2 (en) Fluorescent protein particles
WO2020162203A1 (fr) Mutant enzymatique pour méthode d'immunoessai homogène
Akter et al. Aptamer-based protein detection using a bioluminescent fusion protein
Inouye et al. Purification of histidine-tagged aequorin with a reactive cysteine residue for chemical conjugations and its application for bioluminescent sandwich immunoassays
WO2020241486A1 (fr) Procédé d'immunoessai homogène utilisant un anticorps recombinant auquel a été ajouté un fragment de protéine rapporteur
US20240140995A1 (en) Thermostable affinity polypeptides
Ding et al. The proximity between helix I and helix XI in the melibiose carrier of Escherichia coli as determined by cross-linking
Iwai et al. Creation of Antigen-Dependent β-Lactamase Fusion Protein Tethered by Circularly Permuted Antibody Variable Domains
Yang Expression, purification and biophysical characterization of human Presenilin 2

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 16888194

Country of ref document: EP

Kind code of ref document: A1

ENP Entry into the national phase

Ref document number: 2017563741

Country of ref document: JP

Kind code of ref document: A

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 16888194

Country of ref document: EP

Kind code of ref document: A1