JP2015029472A - Method for measuring protease activity - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for detecting/measuring protease in a biological sample with less steps and shorter operation time than the conventional methods using antibodies, which method would not require accurate molecular designing, would generate less fluorescence or luminescence that is a cause of background, and is free from such a problem that time lag is liable to occur between protease cleavage and luciferase rearrangement.SOLUTION: The method of the invention for measuring protease activity comprises the steps of: preparing a carrier to which a reporter protein is immobilized via a protease-recognizing sequence; bringing the carrier in contact with a protease; collecting the reporter protein released from the carrier after the cleavage of the protease-recognizing sequence by the action of the protease; and determining the luminescence, color development or fluorescence derived from the collected reporter protein. The invention also provides an assay kit for measuring protease activity comprising a carrier on which a reporter protein is immobilized via a protease-recognizing sequence, as well as a carrier on which a reporter protein is immobilized via a protease-recognizing sequence.

Description

  The present invention relates to a method for measuring protease activity.

  Protease is an enzyme that hydrolyzes peptide bonds of peptides (generally less than 100 residues, relatively low molecular weight) and proteins (generally 100 residues or more, relatively high molecular weight) in which amino acids are linked in chains by peptide bonds. It is. Various types of physiologic roles work in a wide range of fields such as nutrient absorption, protein disposal and recycling, biodefense, and activity regulation. Proteases are classified into serine proteases, aspartic proteases (acidic proteases), metalloproteases, cysteine proteases, etc., depending on the catalytic mechanism.

  Measurement of the change in activity of a particular protease is considered significant in the management treatment of a particular disease state. For example, changes in serine prosthesis activity are associated with pathologies such as cancer metastasis, thrombosis, and arthritis. In addition, in viral infections such as HIV and hepatitis C virus, the expression of protease is essential during the maturation process. Therefore, simply detecting their expression plays an important role in infectious disease countermeasures and has public health value.

  In order to recognize a specific protease contained in a sample to date, an immunoassay method using an antibody, an immunoblotting method (Western blotting method), an ELISA method (Enzyme-Linked ImmunoSorbent Assay) and the like are known. Since these methods use antibodies, the procurement of materials becomes expensive. In addition, there is a risk that the antibody will not be recognized due to the frequent occurrence of mutations in viral disease-derived proteases, particularly HIV. Thus, protease detection systems that do not use antibodies have been developed. For example, there is a protease detection probe applied with fluorescence resonance energy transfer (FRET) (Patent No. 4966469 (Patent Document 1), Protein Substrate Set, JPT Peptide Technologies GmbH). This is a phenomenon in which excitation energy moves directly between two adjacent dye molecules (or chromophores) not by electromagnetic waves but by electron resonance. In practice, the N-terminus of the peptide serving as a substrate is labeled with a fluorescent donor substance and the C-terminus is labeled with an acceptor substance. The acceptor substance is a substance that absorbs light called a quencher. Fluorescence generated by exciting the fluorescent donor material is absorbed by the acceptor material by energy transfer. When protease is added, the fluorescent donor substance and the acceptor substance are separated from each other, and FRET does not occur, so that fluorescence can be detected from the fluorescent donor. In this method, a protease recognition sequence can be arbitrarily designed, and detection can be easily performed. However, due to the configuration of the recognition sequence, fluorescence that causes background may be emitted even when the protease is not treated. That is, since it is necessary to design a molecule that precisely calculates the positional relationship between a protease recognition sequence and a fluorescent donor / acceptor, it is not easy to develop and manufacture a detection probe. In addition, a technique based on a change in the structure of a reporter protein, not a fluorescent substance, is also used for detection of protease activity (Special Table 2007-508014 (non-patent document 2)). In this method, a protease recognition sequence is incorporated into luciferase, which is a reporter protein. The luciferase in this state has lost its luminous ability. When protease is added to this and the cleavage occurs, the structure of luciferase returns to its original state, so that it has a light-emitting ability. That is, the presence or absence of this luminescence serves as an index for confirming the presence of protease activity. In this method as well as FRET, the protease recognition sequence can be designed freely. However, the luciferase three-dimensional structure and the recognition sequence may cause steady light emission, and the luciferase shape may not be restored as expected. There is a possibility. Therefore, as in the FRET method, the design of a protease recognition probe requires advanced calculation techniques. In addition, since protein molecules are enormous, reconstitution takes time, and there is a problem that a time lag tends to occur between protease cleavage and luciferase reconstitution, which is not suitable for kinetic analysis.

Patent No. 4966469 Special Table 2007-508014 Publication

  An object of the present invention is to provide a method for detecting and measuring a protease in a biological sample with fewer steps and a shorter working time than a method using a conventional antibody.

  In addition, the present invention does not require precise molecular design, has little fluorescence and luminescence that cause background, and has no problem of time lag between protease cleavage and luciferase reconstitution.・ The purpose is to provide measurement methods.

  As a result of diligent efforts to solve these problems, the inventors have found a new method for measuring protease activity in a biological sample from light emission, color development, and fluorescence derived from a reporter protein, thereby completing the present invention. It came.

The gist of the present invention is as follows.
(1) A step of preparing a carrier on which a reporter protein is immobilized via a protease recognition sequence, a step of bringing a protease into contact with the carrier, a protease recognition sequence is cleaved by the action of the protease, and the reporter protein released from the carrier is recovered. A method for measuring protease activity, comprising a step of measuring the luminescence, color development or fluorescence derived from the recovered reporter protein.
(2) The method according to (1), wherein the carrier is a bead coated with avidin, biotin is bound to the protease recognition sequence, and the reporter protein is immobilized on the carrier by biotin / avidin binding via the protease recognition sequence. .
(3) The carrier is a column packing material in which a metal is immobilized, the His tag is bound to the protease recognition sequence, and the reporter protein is immobilized to the carrier by the His tag / metal bond via the protease recognition sequence ( 1) The method described.
(4) The method according to (3), wherein the metal is selected from the group consisting of nickel, copper, zinc and cobalt.
(5) The support is a plate bottom coated with avidin, biotin is bound to the protease recognition sequence, and the reporter protein is immobilized on the support by biotin / avidin binding via the protease recognition sequence. Method.
(6) The method according to any one of (1) to (5), wherein the protease recognition sequence is a protease recognition sequence of HIV-1.
(7) HIV-1 protease recognition sequence is MA / CA, CA / p2, p2 / NC, NC / p1, p1 / p6 ^gag , NC / TFP, TFP / p6 ^pol , p6 ^pol / PR, PR / RTp51 The method according to (6), wherein the sequence is selected from the group consisting of RT / RTp66, RTp66 / INT and Nef.
(8) The method according to any one of (1) to (7), wherein the reporter protein is luciferase, and the luminescence derived from the reporter protein is luminescence generated by a reaction between luciferase and luciferin.
(9) Prepare a carrier in which different types of reporter proteins are immobilized via different types of protease recognition sequences, and measure the activities of different types of proteases according to (1) to (8) The method according to any one.
(10) A kit for measuring protease activity, comprising a carrier on which a reporter protein is immobilized via a protease recognition sequence.
(11) A carrier on which a reporter protein is immobilized via a protease recognition sequence.
(12) A reporter protein for measuring protease activity, comprising a reporter protein to which a protease recognition sequence is bound.

  According to the present invention, it is possible to detect and measure a protease in a biological sample with fewer steps and a shorter working time than a method using a conventional antibody.

  In addition, according to the present invention, it is possible to detect and measure a protease that does not require precise molecular design, has little fluorescence or luminescence that causes background, and does not cause a time lag between protease cleavage and luciferase reconstitution. It has become possible.

1 is a schematic diagram of an embodiment of the present invention. 1 is a schematic diagram of one embodiment of the present invention. In this embodiment, a reporter protein (luminescent / chromogenic enzyme) is immobilized on a bead (carrier) through an avidin / biotin bond via a protease recognition sequence. 1 is a schematic diagram of one embodiment of the present invention. In this embodiment, a reporter protein (luminescent / chromogenic enzyme) is immobilized on a solid phase carrier by His tag Ni binding via a protease recognition sequence. The structure of the vector produced in Example 1 (biotin / avidin binding system) and Example 2 (His-tag Ni binding system) is shown. Bio: Biotin binding sequence, 3C: Turbo 3C protease recognition sequence, MA / CA: HIV MA / CA sequence, Nc / pl: HIV Nc / pl sequence, Luc: Luciferase, TsLuc: Thermostable luciferase, His: His tag . The detection result (biotin-avidin binding system) of Turbo 3C protease by luminescence of luciferase is shown. The detection result (MA / CA sequence) of HIV-1 protease by luminescence of luciferase (biotin / avidin binding system) is shown. The detection result (Nc / p1 sequence) of HIV-1 protease by luminescence of luciferase (biotin / avidin binding system) is shown. The detection result (MA / CA sequence) of HIV-1 protease by luminescence of luciferase (His-tag Ni binding system) is shown. The detection result (MA / CA sequence) of HIV-1 protease by luminescence of thermostable luciferase (biotin / avidin binding system) is shown. It is a schematic diagram of a method for simultaneously detecting a plurality of viruses in a sample using a carrier in which each of two types of reporter proteins is immobilized via each of two types of protease recognition sequences. FLuc: firefly luciferase, RLuc: Renilla (Renilla) luciferase, GrLuc: green luciferase, RdLuc: red luciferase.

  Hereinafter, embodiments of the present invention will be described in more detail.

  The present invention provides a step of preparing a carrier on which a reporter protein is immobilized via a protease recognition sequence, a step of bringing the carrier into contact with a protease, and a reporter protein released from the carrier by cleavage of the protease recognition sequence by the action of the protease. Provided is a method for measuring protease activity, comprising a step of collecting, and a step of measuring luminescence, color development or fluorescence derived from the collected reporter protein.

  Examples of the protease recognition sequence include, but are not limited to, HIV-1 protease recognition sequence, herpesvirus protease recognition sequence, poliovirus protease recognition sequence, hepatitis C virus protease recognition sequence, and the like. Do not mean.

HIV-1 protease recognition sequences include MA / CA, CA / p2, p2 / NC, NC / p1, p1 / p6 ^gag , NC / TFP, TFP / p6 ^pol , p6 ^pol / PR, PR / RTp51, RT Examples include / RTp66, RTp66 / INT, and Nef, but are not limited thereto.

MA / CA, CA / p2, p2 / NC, NC / p1, p1 / p6 ^gag , NC / TFP, TFP / p6 ^pol , p6 ^pol / PR, PR / RTp51, RT / RTp66, RTp66 / INT, Nef sequence The cleavage sites are as follows.
MA / CA
Ser-Gln-Asn-Tyr / Pro-Ile-Val-Gln (SEQ ID NO: 11)

NC / p1
Arg-Gln-Ala-Asn / Phe-Leu-Gly-Lys (SEQ ID NO: 12)

p2 / NC
Thr-Ala-Ile-Met / Met-Gln-Lys-Gly (SEQ ID NO: 13)
Or
Ser-Ala-Ile-Met / Met-Gln-Lys-Gly (SEQ ID NO: 14)

p1 / p6 ^gag
Pro-Gly-Asn-Phe / Leu-Gln-Ser-Arg (SEQ ID NO: 15)

NC / TFP
Arg-Gln-Ala-Asn / Phe-Leu-Arg-Glu (SEQ ID NO: 16)

TFP / p6 ^pol
Asn-Leu-Ala-Phe / Gln-Gln-Gly-Glu (SEQ ID NO: 17)
Or
Asn-Leu-Ala-Phe / Any-Gln-Gly-Glu (SEQ ID NO: 18)
Or
Asp-Leu-Ala-Phe / Leu-Gln-Gly-Lys (SEQ ID NO: 19)

p6 ^pol / PR
Ser-Phe-Ser-Phe / Pro-Gln-Ile-Thr (SEQ ID NO: 20)
Or
Ser-Phe-Any-Phe / Pro-Gln-Ile-Thr (SEQ ID NO: 21)
Or
Ser-Phe-Asn-Phe / Pro-Gln-Val-Thr (SEQ ID NO: 22)

PR / RTp51
Thr-Leu-Asn-Phe / Pro-Ile-Ser-Pro (SEQ ID NO: 23)

RT / RTp66
Ala-Glu-Thr-Phe / Tyr-Val-Asp-Gly (SEQ ID NO: 24)

RTp66 / INT
Arg-Lys-Val-Leu / Phe-Leu-Asp-Gly (SEQ ID NO: 25)

Nef
Asp-Cys-Ala-Trp / Leu-Glu-Ala-Gln (SEQ ID NO: 26)
Or
Ala-Cys-Ala-Trp / Leu-Glu-Ala-Gln (SEQ ID NO: 27)

CA / p2
Ala-Arg-Val-Leu / Ala-Glu-Ala-Met (SEQ ID NO: 28)

However, the symbols represent the following meanings.
/ Cutting site
Ala alanine
Arg
Asn Asparagine
Asp Aspartic acid
Cys cysteine
Gln Glutamine
Glu glutamic acid
Gly Glycine
Ile isoleucine
Leu leucine
Lys
Met Methionine
Phe phenylalanine
Pro Proline
Ser serine
Thr Threonine
Trp tryptophan
Tyr tyrosine
Val Valine
Any All amino acid reporter proteins include beetle luciferase, Renilla luciferase, Gaussia luciferase, chloramphenicol acetyltransferase (CAT), green fluorescent protein (GFP), peroxidase, alkaline phosphatase, etc. However, the present invention is not limited to these examples.

  The luciferase may be any luciferase that reacts with a luciferase substrate to produce luminescence, and is not particularly limited. Preferably, firefly luciferin derived from a beetle (ie, a multi-complex organic acid D- ( -)-2- (6'hydroxy-2'-benzothiazolyl) -Δ2-thiazoline-4-carboxylic acid) is an enzyme that emits photons by oxidizing it as a luminescent substrate. It includes all enzymes that are derived from luminescent beetles, such as fireflies and Iriomote, and that contribute to the luminescent reaction. This includes enzymes in which the stability (including heat resistance) and luminescence properties of the enzyme protein itself have been artificially modified by recombinant DNA technology or mutation technology. As luciferases having improved thermostability, luciferases disclosed in JP-T-09-510610, JP-T-2008-244507, JP-A-2000-197487, and the like are known, and these may be used.

  When the reporter protein is luciferase, the luminescence generated by the reaction of luciferase and luciferin can be measured as the luminescence derived from the reporter protein. Luminescence generated by the reaction of luciferase and luciferin can be performed by emitting light using a commercially available luciferin / luciferase luminescence reagent (LL luminescence reagent) and measuring the luminescence with a luminometer.

  Examples of the carrier include, but are not limited to, beads, column fillers, plates and the like.

  The beads should be 1-10% highly crosslinked agarose with an average particle size of 50-150um.

  The beads used as the carrier may be magnetic beads. Magnetic beads are convenient because they can be separated magnetically.

  The beads may be used as a column filler.

  As the plate, a microtiter plate can be used. A commercially available microtiter plate can be used. The number of wells includes 6, 24, 96, 384, etc. Any number can be used. As the material, any of acrylic resin, polyethylene, polypropylene, polystyrene, glass and the like can be used.

  The carrier may have a surface on which the reporter protein can be immobilized via a protease recognition sequence. In order to immobilize the reporter protein on the carrier via the protease recognition sequence, biotin / avidin bond, His-tag Ni bond or the like may be used.

  For example, if avidin is coated on the surface of a carrier (eg, bead, plate) and biotin is bound to the protease recognition sequence, the reporter protein can be immobilized on the carrier via the protease recognition sequence by biotin-avidin binding. it can.

  In order to bind biotin to the protease recognition sequence, biotin binding sequence (example of sequence, Leu-Glu-Ala-Met-Lys-Met-Glu-Thr-Glu-Ile (SEQ ID NO: 29) (SEQ ID NO: 29)) Biotin is added to the lysine site by the host that expresses the protein))).

  In addition, if a metal is immobilized on the surface of a carrier (for example, a column packing material) and a His tag is added to the protease recognition sequence, the reporter protein is immobilized on the carrier via the protease recognition sequence by His tag / metal bond. be able to. Examples of the metal include nickel, copper, zinc, and cobalt.

  Furthermore, a His tag may be added to the reporter protein. This makes it possible to purify the reporter protein with a Ni-binding ligand packed column.

  In order to immobilize the reporter protein to the carrier via the protease recognition sequence, as described above, a biotin binding sequence or a His tag sequence may be added to the protease recognition sequence, and the protease recognition sequence to which these sequences are added is bound. A reporter protein (in the examples described later, referred to as “protease recognition probe protein”) may be prepared by a genetic engineering technique, and the protease recognition probe protein may be immobilized on a carrier. When a biotin binding sequence or His tag sequence is added to the N-terminal side of the protease recognition sequence in the protease recognition probe protein, the reporter protein may be bound to the C-terminal side of the protease recognition sequence, and conversely, biotin binding When the sequence or the His tag sequence is added to the C-terminal side of the protease recognition sequence, the reporter protein may be bound to the N-terminal side of the protease recognition sequence. When a protease recognition probe protein is produced by a genetic engineering technique, a vector is used. Therefore, a sequence derived from the vector may be included in the protease recognition probe protein.

  In the method of the present invention, when a protease is brought into contact with a carrier on which a reporter protein is immobilized via a protease recognition sequence, the protease recognition sequence is cleaved by the action of the protease, and the reporter protein is released from the carrier. Protease activity can be measured by collecting the released reporter protein and measuring luminescence, color development or fluorescence derived from the collected reporter protein.

  When the carrier is a bead, the carrier and the protease may be contacted in solution. The protease recognition sequence is cleaved by the action of the protease, and the reporter protein released from the carrier can be recovered from the supernatant by precipitating the carrier by centrifugation. If the carrier is a magnetic bead, the carrier can be precipitated magnetically rather than by centrifugation, and the free reporter protein can be recovered from the supernatant.

  If the carrier is a column packing, a protease-recognizing sequence is cleaved by the action of the protease and a reporter protein released from the carrier flows out if the solution containing the protease flows through the column. The free reporter protein can be recovered.

  The conditions for contacting the protease with the carrier are 20 to 40 ° C., preferably 25 to 30 ° C. and pH 2 to 8. The optimum pH of HIV-1 protease is 2-5.

  Luminescence, color development or fluorescence derived from the collected reporter protein can be measured by a conventional method. For example, if the reporter protein is luciferase, light may be emitted using a commercially available luciferin / luciferase luminescence reagent (LL luminescence reagent) and the luminescence may be measured with a luminometer.

  If the photoprotein is Renilla luciferase, a solution containing 1 mg / ml coelenterazine is added to the collected solution to emit light, and the luminescence is measured with a luminometer.

  When peroxidase is used as a reporter protein, both luminescence and color development can be measured. In other words, since peroxidase is an enzyme that activates oxygen atoms of hydrogen peroxide to oxidize the substrate, using the principle of this enzyme reaction, the amount of the enzyme is determined using a dye that emits light or develops color when oxidized to the substrate. Can be quantified. In the case of luminescence, the luminometer is used, and in the case of color development, the dye concentration is quantified with an absorbance analyzer utilizing the property of the dye that absorbs a specific wavelength.

  When the fluorescent protein is used as the reporter protein, the fluorescence of the protein released by the cleavage reaction of the protease reaction is measured using an instrument that simultaneously performs excitation light irradiation and fluorescence measurement. Specifically, it is a fluorescence spectrophotometer, a microplate reader, or the like.

  In order to verify whether the detected luminescence, color development, or fluorescence is caused by cleavage of the protease, a test section to which a protease inhibitor is added during the protease reaction may be set. Even if luminescence, color development, or fluorescence is detected from a biological sample such as blood, if no luminescence, color development, or fluorescence is detected in the test section where a protease inhibitor is added to the same biological sample, the detected luminescence, color development, or fluorescence is the protease. However, if luminescence, color development or fluorescence was detected even in the test section where the protease inhibitor was added to the same biological sample, the detected luminescence, color development or fluorescence was generated by cleavage of the protease. It cannot be said that it is a thing.

  In the present invention, the reporter protein and the protease recognition sequence immobilized on the carrier are not limited to one type each, and may be a plurality of types. That is, a carrier in which different types of reporter proteins are immobilized via different types of protease recognition sequences may be prepared, and the activities of different types of proteases may be measured. For example, as shown in FIG. 10 (Example 1), one measurement system includes a carrier on which firefly luciferase is immobilized through an HIV protease recognition sequence and a carrier on which renal luciferase is immobilized through a hepatitis C virus recognition sequence. Prepare multiple viruses simultaneously. Alternatively, as shown in FIG. 10 (Example 2), one measurement system consists of a carrier immobilizing green light luciferase via an HIV protease recognition sequence and a carrier immobilizing red light luciferase via a hepatitis C virus recognition sequence. Prepare multiple viruses simultaneously.

  The present invention also provides a kit for measuring protease activity, which comprises a carrier on which a reporter protein is immobilized via a protease recognition sequence. The carrier on which the reporter protein is immobilized via the protease recognition sequence has been described above.

  The measurement kit of the present invention further includes a protease standard reagent, a protease inhibitor, a reagent for luminescence of a reporter protein (for example, luciferin / luciferase luminescence reagent), a reagent solution (for example, a luminescence reagent solution, luminescence, color development, or A calibration curve for associating the amount of fluorescence with the amount of protease, instruction manuals, software for judging negative / positive from the numerical results may be included.

  The method and kit for measuring protease activity of the present invention can be used for the detection and quantification of microorganisms (including viruses) that produce protease.

  Furthermore, the present invention provides a carrier on which a reporter protein is immobilized via a protease recognition sequence. The carrier on which the reporter protein is immobilized via the protease recognition sequence has been described above. A carrier on which a reporter protein is immobilized via a protease recognition sequence can be used for detection and quantification of microorganisms (including viruses) that produce protease.

  Furthermore, the present invention provides a reporter protein for measuring protease activity, comprising a reporter protein to which a protease recognition sequence is bound. As mentioned above, it is better to add a biotin binding sequence or His tag sequence to the protease recognition sequence, and create a reporter protein (protease recognition probe protein) that binds the protease recognition sequence to which these sequences have been added using genetic engineering techniques. If this protease recognition probe protein is immobilized on a carrier (described above), microorganisms (including viruses) that produce protease can be detected and quantified using this carrier.

EXAMPLES Hereinafter, although this invention is demonstrated in detail based on an Example, this invention is not limited to these Examples.
[Example 1] Protease activity detection method 1
Experimental Method a) Construction of protease vector (biotin / avidin binding system) (FIG. 4)
A reporter protein expression vector for measuring protease activity was constructed using immobilization on a carrier by the binding ability of avidin / biotin according to the present invention. As the protease recognition sequence, a Turbo 3C protease recognition sequence, or the MA-CA sequence or Nc / p1 sequence of HIV-1 was adopted. First, Trbo3c protease is an enzyme that cleaves between 6th and 7th using Leu-Glu-Val-Leu-Phe-Gln-Gly-Pro (SEQ ID NO: 30) as a recognition sequence. Used in protein purification systems. The MA-CA sequence is Ser-Gln-Asn-Tyr-Pro-Ile-Val-Gln (SEQ ID NO: 11), and the Nc / p1 sequence is Arg-Gln-Ala-Asn-Phe-Leu-Gly-Lys. (SEQ ID NO: 12). When HIV-1 is budding as an immature virus particle, the HIV-1 protease it contains becomes a mature virus with infectivity by processing the precursor protein Pr55 gag -pol in the virus particle. Of the sequence recognized and cleaved by HIV-1 protease [de Oliveira T et al. Variability at HIV-1 Subtype C Protease Cleavage Sites: An Indication of Viral Fitness? Journal of Virology (2003), 77 (17) : 9422-30]. Note that the cut site exists between the 4th and 5th sequences in any sequence.

  First, in order to add a protease recognition sequence to luciferase, a primer containing a sequence expressing the protease recognition sequence on the 5 ′ end side of luciferase and a primer containing the C terminus of luciferase were synthesized and PCR was performed. Thereby, a gene fragment expressing a protein in which the amino acid sequence Ser-Gln-Asn-Tyr-Pro-Ile-Val-Gln (SEQ ID NO: 11) and luciferase are fused is obtained. Next, the obtained fragment was inserted into PinPoint Xa Vector (Promega). This vector is designed for the purpose of adding a biotin-binding peptide to the inserted sequence. The biotin-binding sequence is added to the fifth lysine site of Leu-Glu-Ala-Met-Lys-Met-Glu-Thr-Glu-Ile (SEQ ID NO: 29) by the host that expresses the protein. This principle is known from the literature [Chapman-Smith, A., and Cronan, J.E. Jr. (1999) J. Nutr. 129: 477S-484S.]. Next, primers were synthesized from the obtained vector to obtain a fragment for inserting a fragment containing a biotin-binding peptide, protease recognition sequence, and luciferase into a protein expression vector, and PCR was performed. The obtained DNA fragment amplified by PCR was inserted into a pTriEx3 Neo vector (Merck). The pTriEx-3 vector enables large-scale expression of the target protein in any of Escherichia coli, yeast, insect cells, and mammalian cells.

b) Expression and purification of reporter protein for measuring protease activity (biotin / avidin binding system)
First, it was confirmed whether the expression vector obtained in a) expresses the target protein. That is, the expression vector was transformed into E. coli BL21 DE3 (pLysS) by a conventional method. E. coli carrying the expression vector has drug resistance (ampicillin). This Escherichia coli was inoculated into 2.5 ml of LB medium containing 1 mg · ml of amplifier and cultured overnight. Next, 100 ul of the culture solution was newly transferred to 2.5 ml of the culture solution and cultured for about 5 hours. The mixture was incubated on ice for 30 minutes, and IPTG was added to a final concentration of 0.1 mM. Thereafter, the cells were cultured at 16 ° C. for 4 hours. In order to easily extract soluble protein from the culture solution, the operation of freezing at -80 ° C for 30 minutes and rapid thawing at 37 ° C was repeated three times to obtain an E. coli disruption solution. This solution was centrifuged at 15000 rpm for 15 minutes, and the supernatant was used as a soluble protein solution. It was assumed that the reporter protein was normally produced in Escherichia coli by separating a part and performing SDS-PAGE to confirm the band at the target position.

  Next, in order to obtain a large amount of high-purity reporter protein, scale-up and purification of the protein using a Ni column were performed. First, Escherichia coli was cultured on a 200 ml scale, and when OD600 reached 0.5, it was allowed to stand on ice for 30 minutes. IPTG was added to a final concentration of 0.1 mM and cultured at 16 ° C. for 4 hours.

  E. coli was recovered by centrifugation, and the resulting E. coli pellet was washed twice with 10 ml of cold PBS. Next, 9 ml of sonication buffer [20 mM Tris-HCl (pH 8.0), 500 mM NaCl, 1 mM EDTA, 1 mM PMSF] was added and sonicated on ice. The treated solution was rapidly frozen at -80 ° C. for 10 minutes, and then the sorbed solid material was sonicated for 30 seconds at room temperature. The above freeze-thaw treatment was repeated 3 times. Subsequently, 1 ml of 10% Triton X-100 was added and shaken at 4 ° C. for 1 hour. In order to remove impurities, the mixture was centrifuged at 12,000 rpm for 10 minutes, and the supernatant was used for purification.

  Next, the His-tag-labeled protein was specifically purified from the soluble total protein obtained by this operation. HiTrap Chelating HP (GE Healthcare) was used for purification. First, a 10 ml syringe was connected to the column, and 5 ml of ultrapure water was fed at a flow rate of 1 drop / second to replace the column stock solution with ultrapure water. Subsequently, 0.5 ml of 0.1M nickel sulfate solution was fed at a flow rate of 1 drop / 2 seconds to bind nickel ions to the ligand. 5 ml of ultrapure water was fed at a flow rate of 1 drop / second to wash away unbound nickel ions from the ligand. In order to equilibrate the column, 10 ml of a binding buffer (20 mM sodium phosphate, 0.5 M sodium chloride, 10 mM imidazole, pH 7.4) was fed at a flow rate of 1 drop / second. Next, the supernatant was sent at a flow rate of 1 drop / 2 seconds to bind the protein containing His-tag to the ligand. In order to remove proteins other than the fungus and the target product, the column was washed by feeding 10 ml of a binding buffer at a flow rate of 1 drop / second. Next, 5 ml of elution buffer [20 mM sodium phosphate, 500 mM NaCl, 500 mM imidazole (pH 7.4) was fed at a flow rate of 1 drop / second, and the eluate was collected. The absorbance of the eluate was measured at 280 nm to calculate the concentration of the target protein, and the yield was calculated. Subsequently, in order to replace the elution buffer with a buffer suitable for the protease reaction, the protein was concentrated by ultrafiltration and the target protein was re-reconstituted with PBS buffer (2.68 mM KCl, 136.89 mM NaCl, 1.47 mM KH2PO4, 8.10 mM Na2HPO4). Dissolved. For ultrafiltration, an Amicon Ultra 30K device (Merck) was used. 500 ml of the eluted sample was put in amicon lutar, centrifuged at 14,000 rpm for 30 minutes, and the protein on the filter was redissolved with 20 ml of PBS buffer to obtain a high concentration probe protein solution.

c) Immobilization of probe protein (biotin / avidin binding system)
In order to bind the probe protein and the solid phase carrier, the following operation was performed. That is, Dyaabeads Myone Streptavidin T1 (Invitrogen) (hereinafter referred to as streptavidin-coated beads), which is a microbead in which the probe protein solution obtained above and streptavidin are coated on the surface of the fine particle metal. The upper limit of biotinylated protein to which streptavidin-coated beads can bind is 20 ug per 100 ul of bead solution. First, the stock solution of streptavidin-coated beads is replaced with PBS buffer. Dispense 100ul of streptavidin-coated bead suspension into tubes. The bottom of the tube is brought close to magnetism to precipitate the beads. Remove the supernatant by aspiration, add 200ul of PBS buffer and mix by inverting to wash the beads. Again, bring the bottom of the tube close to magnetism to precipitate the beads and aspirate the buffer. Repeat this operation three times. Next, the high concentration probe protein solution is added to the streptavidin-coated beads so that the protein amount does not exceed 20 ug. The biotinylated protein is bound to the beads using a microtube rotator by mixing for 30 minutes at room temperature. Then, bring the bottom of the tube close to magnetism to precipitate the beads, remove the supernatant, and resuspend with 100 ul of protease reaction buffer (50 mM Na acetate [pH 5.5], 1 M NaCl, 1 mM dithiothreitol, 1 mM EDTA). It becomes cloudy. This suspension was used as a protease recognition probe solution.

d) Measurement of protease activity (biotin / avidin binding system)
It was examined whether the obtained probe protein can actually measure the activity of a specific protease. 1 unit of Turbo 3C protease for 20ul of protease recognition probe solution with Turbo 3C protease recognition sequence, 1 unit of HIV-1 protease (ANASPEC) for 20ul of protease recognition probe solution with MA-CA or Nc / p1 sequence And allowed to react at room temperature for 15 minutes. The protease was mixed well by tapping every 5 minutes. Magnetic beads were precipitated by bringing the bottom of the tube close to magnetism, and the supernatant was collected in a separate tube. 100 ul of Picker Gene PGL-100 was added to the collected supernatant, and the amount of luminescence for 10 seconds was measured with a luminometer LV9800 (Berthold). In addition, in order to verify whether or not the detected luminescence was caused by cleavage of the protease, a test group was also set in which a protease inhibitor was added simultaneously during the protease reaction.

Experimental results are shown in FIGS.

  FIG. 5 shows the detection result of Turbo 3C protease by luciferase luminescence. Protease (+): The result of luminescence measurement after adding 1 unit of Turbo 3C protease. Protease (-): Without adding Turbo 3C protease. Protease (+) + Inhibitor: Results of luminescence measurement after simultaneously adding 5ul of Turbo 3C protease 1 unit and Protease Inhibitor Coktail (Sigma Aldridge).

  Luminescence was confirmed only in the Protease (+) test group, and luminescence was at the background level in the Protease (+) Inhibitor (+) test group. Inhibitor is a protease inhibitor that specifically inhibits the activity of Turbo3C protease. For this reason, the detected luminescence is luminescence derived from luciferase cleaved and released by the protease.

  FIG. 6 shows the detection result of HIV-1 protease detection result (MA / CA sequence) by luminescence of luciferase. Protease (+): The result of luminescence measurement after adding 1 unit of HIV-1 protease. Protease (-): HIV-1 protease is not added. Protease + Inhibitor: Results of luminescence measurement after simultaneously adding 1 unit of HIV-1 protease and 5ul of Protease Inhibitor Coktail (Sigma Aldridge).

  Luminescence was confirmed only in the Protease (+) test group, and luminescence was at the background level in the Protease (+) Inhibitor (+) test group. Inhibitor is a protease inhibitor that specifically inhibits the activity of HIV-1 protease. For this reason, the detected luminescence is luminescence derived from luciferase cleaved and released by the protease.

  FIG. 7 shows the detection result of the HIV-1 protease detection result (NC / pl sequence) by luminescence of luciferase. Protease (+): Luminescence measurement after adding 1 unit of HIV-1 protease. Protease (-): HIV-1 protease is not added. Protease + Inhibitor: Results of luminescence measurement after simultaneously adding 1 unit of HIV-1 protease and 5ul of Protease Inhibitor Coktail (Sigma Aldridge).

  Luminescence was confirmed only in the Protease (+) test group, and luminescence was at the background level in the Protease (+) Inhibitor (+) test group. Inhibitor is a protease inhibitor that specifically inhibits the activity of HIV-1 protease. For this reason, the detected luminescence is luminescence derived from luciferase cleaved and released by the protease.

[Example 2] Method 2 for detecting protease activity
Experimental method e) Construction of protease vector (His-tag Ni binding system) (Fig. 4)
A protease detection system using only the His-tag as a binding site, independent of the avidin-biotin binding system, was examined. First, in order to add the MA / CA sequence, which is the HIV-1 protease recognition sequence, to luciferase, a primer containing a sequence that expresses the protease recognition sequence on the 5 ′ end side of luciferase and a primer containing the C terminus of luciferase were synthesized. PCR was performed. Thereby, a gene fragment expressing a protein in which the amino acid sequence Ser-Gln-Asn-Tyr-Pro-Ile-Val-Gln (SEQ ID NO: 11) and luciferase are fused is obtained. The obtained DNA fragment amplified by PCR was inserted into a pTriEx3 Neo vector (Merck). The pTriEx-3 vector enables large-scale expression of the target protein in any of Escherichia coli, yeast, insect cells, and mammalian cells. This vector also adds a His-tag to the C-terminus of the protein to be expressed.

f) Expression and purification of reporter protein for measuring protease activity (His-tag Ni binding system)
First, it was confirmed whether the expression vector obtained in e) expresses the target protein. That is, the expression vector was transformed into E. coli BL21 DE3 (pLysS) by a conventional method. E. coli carrying the expression vector has drug resistance (ampicillin). This Escherichia coli was inoculated into 2.5 ml of LB medium containing 1 mg · ml of amplifier and cultured overnight. Next, 100 ul of the culture solution was newly transferred to 2.5 ml of the culture solution and cultured for about 5 hours. The mixture was incubated on ice for 30 minutes, and IPTG was added to a final concentration of 0.1 mM. Thereafter, the cells were cultured at 16 ° C. for 4 hours. In order to easily extract soluble protein from the culture solution, the operation of freezing at -80 ° C for 30 minutes and rapid thawing at 37 ° C was repeated three times to obtain an E. coli disruption solution. This solution was centrifuged at 15000 rpm for 15 minutes, and the supernatant was used as a soluble protein solution. It was assumed that the reporter protein was normally produced in Escherichia coli by separating a part and performing SDS-PAGE to confirm the band at the target position.

  Next, in order to obtain a large amount of high-purity reporter protein, scale-up and purification of the protein using a Ni column were performed. First, Escherichia coli was cultured on a 200 ml scale, and when OD600 reached 0.5, it was allowed to stand on ice for 30 minutes. IPTG was added to a final concentration of 0.1 mM and cultured at 16 ° C. for 4 hours.

  E. coli was recovered by centrifugation, and the resulting E. coli pellet was washed twice with 10 ml of cold PBS. Next, 9 ml of sonication buffer [20 mM Tris-Cl (pH 8.0), 500 mM NaCl, 1 mM EDTA, 1 mM PMSF] was added and sonicated on ice. The treated solution was rapidly frozen at -80 ° C. for 10 minutes, and then the sorbed solid material was sonicated for 30 seconds at room temperature. The above freeze-thaw treatment was repeated 3 times. Subsequently, 1 ml of 10% Triton X-100 was added and shaken at 4 ° C. for 1 hour. In order to remove impurities, the mixture was centrifuged at 12,000 rpm for 10 minutes, and the supernatant was used for purification.

  Next, the His-tag-labeled protein was specifically purified from the soluble total protein obtained by this operation. HiTrap Chelating HP (GE Healthcare) was used for purification. First, a 10 ml syringe was connected to the column, and 5 ml of ultrapure water was fed at a flow rate of 1 drop / second to replace the column stock solution with ultrapure water. Subsequently, 0.5 ml of 0.1M nickel sulfate solution was fed at a flow rate of 1 drop / 2 seconds to bind nickel ions to the ligand. 5 ml of ultrapure water was fed at a flow rate of 1 drop / second to wash away unbound nickel ions from the ligand. In order to equilibrate the column, 10 ml of a binding buffer (20 mM sodium phosphate, 0.5 M sodium chloride, 10 mM imidazole, pH 7.4) was fed at a flow rate of 1 drop / second. Next, the supernatant was sent at a flow rate of 1 drop / 2 seconds to bind the protein containing His-tag to the ligand. In order to remove proteins other than the fungus and the target product, the column was washed by feeding 10 ml of a binding buffer at a flow rate of 1 drop / second. Next, 5 ml of an elution buffer [20 mM sodium phosphate, 500 mM NaCl, 500 mM imidazole (pH 7.4) was fed at a flow rate of 1 drop / second, and the eluate was collected. The absorbance of the eluate was measured at 280 nm to calculate the concentration of the target protein, and the yield was calculated. Subsequently, in order to replace the elution buffer with a buffer suitable for the protease reaction, the protein was concentrated by ultrafiltration and the target protein was re-reconstituted with PBS buffer (2.68 mM KCl, 136.89 mM NaCl, 1.47 mM KH2PO4, 8.10 mM Na2HPO4). Dissolved. For ultrafiltration, an Amicon Ultra 30K device (Merck) was used. 500 ml of the eluted sample was put in amicon lutar, centrifuged at 14,000 rpm for 30 minutes, and the protein on the filter was redissolved with 20 ml of PBS buffer to obtain a high concentration probe protein solution.

g) Immobilization of probe protein (His-tag Ni binding system)
In order to bind the probe protein and the solid phase carrier, the following operation was performed. That is, in the probe protein solution obtained above and agarose, by mixing metal chelate agarose (Wako Pure Chemicals) (hereinafter referred to as Ni beads), which is a microbead in which nickel ions are chelated by iminodiacetic acid, The His-tag in the probe protein is bound to the nickel ion of the Ni beads. The protein to which His-tag is added is 20 mg per ml of agarose bead solution. First, in order to replace the Ni bead stock solution with PBS buffer, 100 ul of Ni bead solution was dispensed into tubes. Allow the tube to settle to allow the beads to settle. Carefully remove the supernatant, add 200ul of PBS buffer and mix by inverting to wash the beads. The tube is allowed to stand again to precipitate the beads, and the PBS buffer is removed by suction. Repeat this operation three times. Next, the high concentration probe protein solution is added to the Ni beads so that the protein amount does not exceed 2 mg. Using a microtube rotator, the His-tag labeled probe protein is bound to the Ni beads by mixing for 30 minutes at room temperature. Then, let the tube stand to precipitate the beads, remove the supernatant, and resuspend in 100 ul of protease reaction buffer (50 mM Na acetate [pH 5.5], 1 M NaCl, 1 mM dithiothreitol, 1 mM EDTA). . This suspension was used as a protease recognition probe solution.

h) Measurement of protease activity (His-tag Ni binding system)
It was examined whether the obtained probe protein can actually measure the activity of a specific protease. One unit of HIV-1 protease (ANASPEC) was added to 20 ul of protease recognition probe solution having the MA-CA sequence and allowed to react at room temperature for 15 minutes. The protease was mixed well by tapping every 5 minutes. Thereafter, the tube was lightly centrifuged to precipitate the beads, and the supernatant was collected in another tube. 100 ul of Picker Gene PGL-100 was added to the collected supernatant, and the amount of luminescence for 10 seconds was measured with a luminometer LV9800 (Berthold). In addition, in order to verify whether or not the detected luminescence was caused by cleavage of the protease, a test group was also set in which a protease inhibitor was added simultaneously during the protease reaction.

The experimental result is shown in FIG.
FIG. 8 shows the detection result of HIV-1 protease detection result (MA / CA sequence) by luciferase luminescence. Protease (+): The result of luminescence measurement after adding 1 unit of HIV-1 protease. Protease (-): HIV-1 protease is not added. Protease + Inhibitor: Results of luminescence measurement after simultaneously adding 1 unit of HIV-1 protease and 5ul of Protease Inhibitor Coktail (Sigma Aldridge).

  Luminescence was confirmed only in the Protease (+) test group, and luminescence was at the background level in the Protease (+) Inhibitor (+) test group. Inhibitor is a protease inhibitor that specifically inhibits the activity of HIV-1 protease. For this reason, the detected luminescence is luminescence derived from luciferase cleaved and released by the protease.

[Example 3] Protease activity detection method 3
Example 1 except that thermostable luciferase (Japanese National Standard Publication No. 09-510610, so-called E354K) was used as a reporter protein, and the reaction between HIV-1 protease and a protease recognition probe having an MA-CA sequence was carried out at 30 ° C. The experiment was carried out in the same manner as described above. The results are shown in FIG.

  FIG. 9 shows the detection results of HIV-1 protease detection results (MA / CA sequence) by thermoluminescence of thermostable luciferase. Protease (+): The result of luminescence measurement after adding 1 unit of HIV-1 protease. Protease (-): HIV-1 protease is not added. Protease + Inhibitor: Results of luminescence measurement after simultaneously adding 1 unit of HIV-1 protease and 5ul of Protease Inhibitor Coktail (Sigma Aldridge).

  Luminescence was confirmed only in the Protease (+) test group, and luminescence was at the background level in the Protease (+) Inhibitor (+) test group. Inhibitor is a protease inhibitor that specifically inhibits the activity of HIV-1 protease. For this reason, the detected luminescence is luminescence derived from luciferase cleaved and released by the protease. The light emission in the Protease (+) test section is about 100 times the light emission shown in Example 1. This is considered to be because resistance is increased when thermostable luciferase is used against operations that interfere with luciferase stability, such as a binding operation to a carrier or a protease reaction.

  According to the present invention, protease can be detected and measured with a small number of steps and a short working time.

  According to the present invention, it is possible to detect and measure a protease that does not require precise molecular design, has little fluorescence and luminescence that cause background, and does not cause a time lag between protease cleavage and luciferase reconstitution. Become.

  The present invention can also be used for detection and quantification of microorganisms that produce protease.

<SEQ ID NO: 1> This shows the DNA sequence of the DNA fragment (Bio-Turbo3C-Luc-His) inserted into the pTriEx3 Neo vector in Example 1a) and the encoded amino acid sequence (three-letter code).
Nucleotide number 1-252: Sequence derived from PinPoint Xa Vector (Promega) Nucleotide number 253-282: Bio (biotin binding sequence) coding region Nucleotide number 283-384: Sequence derived from PinPoint Xa Vector (Promega) Nucleotide number 385-408: 3C (Turbo 3C protease recognition sequence) coding region nucleotide number 409-426: Sequence containing restriction enzyme site nucleotide number 427-2064: Luc (luciferase) coding region nucleotide number 2064-2142: Sequence nucleotide derived from pTriEx3 Neo vector (Merck) No. 2143-2166: His (His tag) coding region nucleotide number 2167-2169: DNA fragment (Bio-Turbo3C-Luc-His) inserted into the pTriEx3 Neo vector at the codon <SEQ ID NO: 2> Example 1a) is encoded Amino acid sequence is shown.
<SEQ ID NO: 3> This shows the DNA sequence of the DNA fragment (Bio-MA / CA-Luc-His) inserted into the pTriEx3 Neo vector in Example 1a) and the encoded amino acid sequence (three-letter code).
Nucleotide number 1-252: Sequence derived from PinPoint Xa Vector (Promega) Nucleotide number 253-282: Bio (biotin binding sequence) coding region Nucleotide number 283-384: Sequence derived from PinPoint Xa Vector (Promega) Nucleotide number 385-408: MA / CA (HIV-1 protease recognition sequence MA / CA) coding region nucleotide number 409-426: sequence containing restriction enzyme site nucleotide number 427-2064: Luc (luciferase) coding region nucleotide number 2064-2142: pTriEx3 Neo vector (Merck) -derived sequence nucleotide number 2143-2166: His (His tag) coding region nucleotide number 2167-2169: stop codon <SEQ ID NO: 4> DNA fragment inserted into pTriEx3 Neo vector in Example 1a) (Bio-MA / The amino acid sequence encoded by (CA-Luc-His) is shown.
<SEQ ID NO: 5> This shows the DNA sequence of the DNA fragment (Bio-Nc / p1-Luc-His) inserted into the pTriEx3 Neo vector in Example 1a) and the encoded amino acid sequence (three-letter code).
Nucleotide number 1-252: Sequence derived from PinPoint Xa Vector (Promega) Nucleotide number 253-282: Bio (biotin binding sequence) coding region Nucleotide number 283-384: Sequence derived from PinPoint Xa Vector (Promega) Nucleotide number 385-408: Nc / p1 (HIV-1 protease recognition sequence Nc / p1) coding region nucleotide number 409-426: sequence containing restriction enzyme site nucleotide number 427-2064: Luc (luciferase) coding region nucleotide number 2064-2142: pTriEx3 Neo vector (Merck) -derived sequence nucleotide number 2143-2166: His (His tag) coding region nucleotide number 2167-2169: stop codon <SEQ ID NO: 6> DNA fragment inserted into pTriEx3 Neo vector in Example 1a) (Bio-Nc / p1-Luc-His) shows the amino acid sequence encoded.
<SEQ ID NO: 7> This shows the DNA sequence of the DNA fragment (Luc-MA / CA-His) inserted into the pTriEx3 Neo vector in Example 2e) and the encoded amino acid sequence (three-letter code).
Nucleotide number 1-33: Sequence derived from pTriEx3 Neo vector (Merck) Nucleotide number 34-1671: Luc (luciferase) coding region Nucleotide number 1672-1689: Sequence containing restriction enzyme sites Nucleotide number 1690-1713: MA / CA (HIV -1 protease recognition sequence MA / CA) coding region nucleotide number 1714-1773: sequence derived from pTriEx3 Neo vector (Merck) nucleotide number 1774-1797: His (His tag) coding region nucleotide number 1798-1800: stop codon <sequence No. 8> shows the amino acid sequence encoded by the DNA fragment (Luc-MA / CA-His) inserted into the pTriEx3 Neo vector in Example 2e).
<SEQ ID NO: 9> This shows the DNA sequence of the DNA fragment (Bio-MA / CA-TsLuc-His) inserted in pTriEx3 Neo vector in Example 3 and the encoded amino acid sequence (three-letter code).
Nucleotide number 1-252: Sequence derived from PinPoint Xa Vector (Promega) Nucleotide number 253-282: Bio (biotin binding sequence) coding region Nucleotide number 283-384: Sequence derived from PinPoint Xa Vector (Promega) Nucleotide number 385-408: MA / CA (HIV-1 protease recognition sequence MA / CA) coding region nucleotide number 409-426: sequence containing restriction enzyme site nucleotide number 427-2064: TsLuc (heat-stable luciferase) coding region * nucleotide numbers 1495-1497 Replaced with aaa (Lys).
Nucleotide number 2064-2142: Sequence derived from pTriEx3 Neo vector (Merck) Nucleotide number 2143-2166: His (His tag) Coding region nucleotide number 2167-2169: Stop codon <SEQ ID NO: 10> Inserted into pTriEx3 Neo vector in Example 3 Shows the amino acid sequence encoded by the DNA fragment (Bio-MA / CA-TsLuc-His).
<SEQ ID NO: 11> Shows the MA / CA sequence.
<SEQ ID NO: 12> Shows the NC / p1 sequence.
<SEQ ID NO: 13> This shows the p2 / NC sequence.
<SEQ ID NO: 14> This shows the p2 / NC sequence.
<SEQ ID NO: 15> This shows the p1 / p6 ^gag sequence.
<SEQ ID NO: 16> Shows the NC / TFP sequence.
<SEQ ID NO: 17> This shows the TFP / p6 ^pol sequence.
<SEQ ID NO: 18> This shows the TFP / p6 ^pol sequence.
<SEQ ID NO: 19> This shows the TFP / p6 ^pol sequence.
<SEQ ID NO: 20> This shows the p6 ^pol / PR sequence.
<SEQ ID NO: 21> This shows the p6 ^pol / PR sequence.
<SEQ ID NO: 22> This shows the p6 ^pol / PR sequence.
<SEQ ID NO: 23> This shows the PR / RTp51 sequence.
<SEQ ID NO: 24> This shows the RT / RTp66 sequence.
<SEQ ID NO: 25> This shows the RTp66 / INT sequence.
<SEQ ID NO: 26> This shows the Nef sequence.
<SEQ ID NO: 27> This shows the Nef sequence.
<SEQ ID NO: 28> This shows the CA / p2 sequence.
<SEQ ID NO: 29> This shows the biotin-binding sequence.
<SEQ ID NO: 30> This shows the recognition sequence of Trbo3c protease (cleaved between the 6th and 7th positions).

Claims (12)

A step of preparing a carrier on which a reporter protein is immobilized via a protease recognition sequence, a step of bringing a protease into contact with the carrier, a step of recovering a reporter protein released from the carrier by cleavage of the protease recognition sequence by the action of the protease, A method for measuring protease activity, comprising a step of measuring luminescence, color development or fluorescence derived from a collected reporter protein. The method according to claim 1, wherein the carrier is beads coated with avidin, biotin is bound to the protease recognition sequence, and the reporter protein is immobilized on the carrier by biotin / avidin binding via the protease recognition sequence. The carrier is a column packing material on which a metal is immobilized, a His tag is bound to a protease recognition sequence, and a reporter protein is immobilized on the carrier by a His tag / metal bond via the protease recognition sequence. the method of. The method of claim 3 wherein the metal is selected from the group consisting of nickel, copper, zinc and cobalt. The method according to claim 1, wherein the carrier is the bottom of the plate coated with avidin, biotin is bound to the protease recognition sequence, and the reporter protein is immobilized on the carrier by biotin / avidin binding via the protease recognition sequence. The method according to any one of claims 1 to 5, wherein the protease recognition sequence is a protease recognition sequence of HIV-1. HIV-1 protease recognition sequence is MA / CA, CA / p2, p2 / NC, NC / p1, p1 / p6 ^gag , NC / TFP, TFP / p6 ^pol , p6 ^pol / PR, PR / RTp51, RT / The method according to claim 6, wherein the sequence is selected from the group consisting of RTp66, RTp66 / INT and Nef. The method according to any one of claims 1 to 7, wherein the reporter protein is luciferase, and the luminescence derived from the reporter protein is luminescence generated by a reaction between luciferase and luciferin. 9. The activity of each of different types of proteases is measured by preparing a carrier in which each of different types of reporter proteins is immobilized via each of different types of protease recognition sequences. Method. A kit for measuring protease activity, comprising a carrier on which a reporter protein is immobilized via a protease recognition sequence. A carrier on which a reporter protein is immobilized via a protease recognition sequence. A reporter protein for measuring protease activity, comprising a reporter protein to which a protease recognition sequence is bound.