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WO2016051752A1 - Substrat peptidique pour protéase et procédé de mesure de l'activité protéase - Google Patents

Substrat peptidique pour protéase et procédé de mesure de l'activité protéase Download PDF

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WO2016051752A1
WO2016051752A1 PCT/JP2015/004901 JP2015004901W WO2016051752A1 WO 2016051752 A1 WO2016051752 A1 WO 2016051752A1 JP 2015004901 W JP2015004901 W JP 2015004901W WO 2016051752 A1 WO2016051752 A1 WO 2016051752A1
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glycine
amino acid
protease
substrate peptide
gly
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PCT/JP2015/004901
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English (en)
Japanese (ja)
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昌史 後藤
前田　浩
和隆 村山
洋平 山形
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国立大学法人東北大学
国立大学法人東京農工大学
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Priority to JP2016551530A priority Critical patent/JP6497683B2/ja
Publication of WO2016051752A1 publication Critical patent/WO2016051752A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
    • 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
    • C12Q1/37—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase involving peptidase or proteinase
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
    • Y02P20/00—Technologies relating to chemical industry
    • Y02P20/50—Improvements relating to the production of bulk chemicals
    • Y02P20/55—Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups

Definitions

  • the present invention relates to a protease substrate peptide and a method for measuring protease activity using the same. More specifically, the present invention relates to a substrate peptide that is specifically cleaved by a neutral protease derived from Clostridium histolyticum.
  • the living tissue is treated with a proteolytic enzyme (protease).
  • proteolytic enzyme for example, for islet transplantation, pancreatic tissue is treated with collagenase to separate the islets.
  • pancreatic tissue is treated with collagenase to separate the islets.
  • Clostridium histolyticus As an enzyme composition used for separation of cells and the like, a composition containing collagenase produced by Clostridium histolyticum is widely used (see Patent Document 1).
  • Clostridium histolyticus has two collagenases, collagenase I (ColG) and collagenase II (ColH), cysteine endopeptidase, Clostripain (CP), and metalloendopeptidase, Clostridium neutral protease (Neuroprotease): NP) of four proteases.
  • the enzyme composition contains NP and CP in addition to the two types of collagenases.
  • the amount of each protease contained in the enzyme composition derived from Clostridium histolyticus affects the separation efficiency of the cells and the like and the state of the cells after the separation by the composition (see Non-Patent Document 1). . For this reason, measuring the amount of each protease in the enzyme composition in advance is useful for ensuring the uniformity of the enzyme composition and the reproducibility of the separation operation.
  • the amount of protease is generally measured using a substrate for activity measurement.
  • a substrate for measuring the activity of collagenase peptides such as “N- [3- (2-furyl) acroyl] -Leu-Gly-Pro-Ala” are commercially available (Sigma Aldrich, F5135).
  • a substrate that can be cleaved only by the protease to be measured.
  • a substrate peptide specific for CP “Bz-L-Arg-pNA” (Benzoyl-L-arginine-p-nitroanilide) is known. It has been clarified that CP has a high substrate specificity and does not exhibit a cleavage activity for peptides not containing Arg or Lys (see Non-Patent Document 2).
  • the main object of the present invention is to provide a substrate that is specifically cleaved by a neutral protease produced by Clostridium histolyticus.
  • the present invention provides the following [1] to [25].
  • [1] Gly-Xaa-Yaa (Xaa is any one amino acid selected from Phe, Leu, Ile and Trp, Yaa is any one amino acid selected from Tyr, His, Ser and Gly)
  • a protease substrate peptide comprising an amino acid sequence.
  • the substrate peptide of [1] comprising a protecting group that binds to the N-terminus and / or C-terminus of the amino acid sequence.
  • [5] Gly-Xaa-Yaa (Xaa is any one amino acid selected from Phe, Leu, Ile and Trp, Yaa is any one amino acid selected from Tyr, His, Ser and Gly) A peptide comprising an amino acid sequence.
  • the peptide according to any one of [5] to [7] which is specifically cleaved by a neutral protease derived from Clostridium histolyticum.
  • Glycine-phenylalanine-tyrosine Glycine-phenylalanine-histidine. Glycine-phenylalanine-serine. Glycine-phenylalanine-glycine. Glycine-leucine-tyrosine. Glycine-histidine. Glycine-leucine-serine. Glycine-leucine-glycine. Glycine-isoleucine-tyrosine. Glycine-isoleucine-histidine. Glycine-isoleucine-serine. Glycine-isoleucine-glycine. Glycine-tryptophan-tyrosine. Glycine-tryptophan-histidine. Glycine-tryptophan-serine. Glycine-tryptophan-glycine. Glycine-tryptophan-tyrosine. Glycine-tryptophan-histidine. Glycine-tryptophan-serine. Glycine-trypto
  • a method for measuring the activity of a target protease An amino acid sequence of Gly-Xaa-Yaa (where Xaa is any one amino acid selected from Phe, Leu, Ile and Trp, Yaa is any one amino acid selected from Tyr, His, Ser and Gly) A substrate peptide comprising, The protease; The steps of mixing and A procedure for quantifying the cleavage amount of the substrate peptide; Including methods.
  • the substrate peptide comprises a protecting group bonded to the N-terminus and / or C-terminus of the amino acid sequence.
  • Glycine-phenylalanine-tyrosine glycine-phenylalanine-histidine, glycine-phenylalanine-serine, glycine-phenylalanine-glycine, glycine-leucine-tyrosine, glycine-leucine-histidine, glycine-leucine-serine, glycine-leucine-glycine, glycine- Isoleucine-tyrosine, glycine-isoleucine-histidine, glycine-isoleucine-serine, glycine-isoleucine-glycine, glycine-tryptophan-tyrosine, glycine-tryptophan-histidine, glycine-tryptophan-serine, or glycine-tryptophan-glycine.
  • a method for determining the amount of a particular protease contained in a composition comprising: An amino acid sequence of Gly-Xaa-Yaa (where Xaa is any one amino acid selected from Phe, Leu, Ile and Trp, Yaa is any one amino acid selected from Tyr, His, Ser and Gly) A substrate peptide comprising, The composition; The steps of mixing and A procedure for quantifying the cleavage amount of the substrate peptide; Including methods. [17] The method according to [16], wherein the substrate peptide comprises a protecting group bonded to the N-terminus and / or C-terminus of the amino acid sequence.
  • amino acid sequence of the substrate peptide is any one of the following: Glycine-phenylalanine-tyrosine, glycine-phenylalanine-histidine, glycine-phenylalanine-serine, glycine-phenylalanine-glycine, glycine-leucine-tyrosine, glycine-histidine, glycine-leucine-serine, glycine-leucine-glycine, glycine- Isoleucine-tyrosine, glycine-isoleucine-histidine, glycine-isoleucine-serine, glycine-isoleucine-glycine, glycine-tryptophan-tyrosine, glycine-tryptophan-histidine, glycine-tryptophan-serine, or glycine-
  • a method for detecting the presence or absence of a specific protease in a composition An amino acid sequence of Gly-Xaa-Yaa (where Xaa is any one amino acid selected from Phe, Leu, Ile and Trp, Yaa is any one amino acid selected from Tyr, His, Ser and Gly) A substrate peptide comprising, The composition; The steps of mixing and A procedure for detecting cleavage of the substrate peptide; Including methods. [22] The method according to [21], wherein the substrate peptide comprises a protecting group bonded to the N-terminus and / or C-terminus of the amino acid sequence.
  • Glycine-phenylalanine-tyrosine glycine-phenylalanine-histidine, glycine-phenylalanine-serine, glycine-phenylalanine-glycine, glycine-leucine-tyrosine, glycine-leucine-histidine, glycine-leucine-serine, glycine-leucine-glycine, glycine- Isoleucine-tyrosine, glycine-isoleucine-histidine, glycine-isoleucine-serine, glycine-isoleucine-glycine, glycine-tryptophan-tyrosine, glycine-tryptophan-histidine, glycine-tryptophan-serine, or glycine-tryptophan-glycine.
  • the present invention provides a substrate that is specifically cleaved by a neutral protease produced by Clostridium histolyticus.
  • substrate peptide of the protease according to the present invention is Gly-Xaa-Yaa (Xaa is any one amino acid selected from Phe, Leu, Ile and Trp, Yaa is selected from Tyr, His, Ser and Gly) The amino acid sequence of any one of the amino acids).
  • the inventors of the present invention provide Gly-Xaa-Yaa (Xaa is any one amino acid selected from Phe, Leu, Ile and Trp, Yaa is any one amino acid selected from Tyr, His, Ser and Gly.
  • the peptide containing the amino acid sequence of (shown) was found to be specifically cleaved between Gly and Xaa of the amino acid sequence by a neutral protease (NP) produced by Clostridium histolyticum. (See Examples).
  • NP neutral protease
  • cleavage for a peptide is used synonymously with “hydrolysis”.
  • cleaved means that it is not cleaved at all by proteases other than NP, or at least cleaving cannot be detected by techniques well known in the art.
  • Specifically cleaved by NP means, for example, a peptide fragment generated by hydrolysis of a substrate peptide by a protease other than NP with respect to the amount of peptide fragment generated by hydrolysis of the substrate peptide by NP Is 5% or less, preferably 1% or less, more preferably 0.5% or less, and still more preferably 0.1% or less.
  • the substrate peptide according to the present invention preferably has a protecting group at the N-terminal and / or C-terminal in order to enable optical detection of cleavage by protease and / or to prevent digestion by exopeptidase.
  • the protecting group is not particularly limited. For example, 3- (2-furyl) acryloyl group (FA group), benzyloxycarbonyl group, benzoyl group, t-butoxycarbonyl group, acetyl group, succinyl group, 9-fluorenyl A methoxycarbonyl group, an amide group or the like is used.
  • F-Gly when an FA group is added to the N-terminus, “FA-Gly” has an absorption at a wavelength of 334 nm.
  • a 2- (N-methylamino) benzoyl group or 7-methoxycoumarin is used as the fluorescent substance.
  • 2,4-Dinitrophenyl (7-methoxycoumarin-4-yl) acetyl) can be used as a quencher.
  • the length of the substrate peptide according to the present invention is not particularly limited, but is 3 amino acids or more in order to ensure substrate specificity for NP.
  • the substrate peptide according to the present invention is modified with a fluorescent substance or a chromophore, or added with a functional group that causes a change in absorbance with the cleavage of the substrate peptide, as long as the substrate specificity to NP can be maintained. May be.
  • the quenching substance bonded to the N-terminal or C-terminal can be positioned in close proximity to the fluorescent substance bonded to multiple ends, and the substrate can be prevented from exciting the fluorescent substance. It is necessary to design the amino acid sequence length of the peptide.
  • the substrate peptide according to the present invention includes any one or more of the following amino acid sequences.
  • NP for substrate peptides, those containing any one or more of the following amino acid sequences are more preferred.
  • Glycine-phenylalanine-tyrosine Glycine-leucine-tyrosine.
  • Specific examples of the substrate peptide according to the present invention include the following. N- [3- (2-Furyl) acryloyl] -glycine-phenylalanine-tyrosine amide. N- [3- (2-Furyl) acryloyl] -glycine-phenylalanine-histidine amide. N- [3- (2-furyl) acryloyl] -glycine-phenylalanine-serine amide. N- [3- (2-Furyl) acryloyl] -glycine-phenylalanine-glycine amide. N- [3- (2-Furyl) acryloyl] -glycine-leucine-tyrosine amide.
  • the substrate peptide according to the present invention can be used for measuring protease activity. Since the substrate peptide according to the present invention is a substrate specific for NP, it is preferably used for measuring the activity of NP.
  • the method for measuring protease activity according to the present invention includes a procedure for mixing the target protease and the substrate peptide according to the present invention, and a procedure for quantifying the cleavage amount of the substrate peptide.
  • the mixing procedure can be performed by dissolving and reacting the protease and the substrate peptide in a suitable solvent. Conditions such as solvent, protease and substrate peptide concentrations, time, and temperature during the reaction may be set in the same manner as in the conventionally known protease activity measurement.
  • the quantitative procedure can be performed by analyzing the reaction solution obtained by the mixing procedure by liquid chromatography and a UV detector, and detecting and quantifying the generated peptide fragments. Detection and quantification of the generated peptide fragment can also be performed by a conventionally known method such as measuring the absorbance of the substrate peptide and detecting a change in absorbance caused by cleavage by a protease.
  • the enzyme activity value can be calculated from the cleavage amount of the substrate peptide obtained by the quantification procedure.
  • the manufactured NP and the enzyme composition containing NP it can be used for quality control of NP products by calculating the enzyme activity value of NP for each product or for each production lot.
  • the substrate peptide according to the present invention can be used for detecting the presence or absence of a protease in the composition. Since the substrate peptide according to the present invention is specifically cleaved by NP, it is suitably used for detecting whether or not NP is contained in the composition.
  • the composition to be a sample is not particularly limited as long as it may contain NP, but particularly preferably a proteolytic enzyme composition derived from Clostridium histolyticus. It is said.
  • the substrate peptide according to the present invention is not cleaved by collagenase G (ColG), collagenase H (ColH) and clostripain (CP) derived from Clostridium histolyticus, but is specifically cleaved by NP.
  • Such a method for detecting a protease can be applied to detect the presence or absence of NP in a proteolytic enzyme composition derived from Clostridium histolyticus.
  • the proteolytic enzyme composition derived from Clostridium histolyticus broadly includes bacterial cells, crushed bacterial cells, culture supernatants or extracts or purified products thereof, which contain proteases produced by the same. Can be.
  • the method for detecting a protease according to the present invention includes a procedure for mixing the target composition and the substrate peptide according to the present invention, and a procedure for detecting cleavage of the substrate peptide.
  • the mixing procedure may be performed in the same manner as the method for measuring protease activity described above.
  • the detection procedure may be performed according to the quantitative procedure of the method for measuring protease activity described above.
  • the substrate peptide according to the present invention can also be used for determining the amount of protease contained in a composition. Since the substrate peptide according to the present invention is a substrate specific for NP, it is preferably used for quantification of NP contained in the composition.
  • the method for quantifying a protease according to the present invention includes a procedure for mixing the target composition and the substrate peptide according to the present invention, and a procedure for quantifying the cleavage amount of the substrate peptide.
  • the mixing procedure and the quantitative procedure may be performed in the same manner as the method for measuring protease activity described above.
  • the enzyme content can be calculated from the cleavage amount of the substrate peptide in the quantification procedure.
  • proteolytic enzyme composition derived from Clostridium histolyticus is widely used as an enzyme composition used for separating cells and the like.
  • This proteolytic enzyme composition contains ColG, ColH, CP and NP produced by Clostridium histolyticus.
  • the separation efficiency of the cells and the state of the cells after the separation are determined by each protease in the composition. Influenced by quantity.
  • the content of NP that can optimize the separation efficiency of cells and the state of separated cells and the like can be determined.
  • the method for quantifying proteases in the composition according to the present invention when quantification of proteases such as ColG, ColH and CP is performed in addition to NP, “N- [3- (2-furyl)” is used. Acroyl] -Leu-Gly-Pro-Ala ”and“ Bz-L-Arg-pNA ”and the like may be performed using a conventionally known substrate peptide.
  • the purified NP or NP activity can be supplemented.
  • Other proteases eg, thermolysin
  • thermolysin can be added until the optimal NP content or optimal protease content is reached.
  • ⁇ Test Example 1 Degradation of synthetic peptide by NP> Synthesis of pentamer peptides “Tyr-Gly-Phe-Tyr-Glu” (SEQ ID NO: 1) and “Tyr-Gly-Phe-Tyr-Arg” (SEQ ID NO: 2) as candidate substrate peptides specific for NP did.
  • cleavage by NP see Reference Example 1
  • collagenase G ColG, Meiji Seika Pharma Co., Ltd.
  • collagenase H ColH, Meiji Seika Pharma Co., Ltd.
  • Clostridium histolyticus The resulting peptide fragments were analyzed by the following method.
  • Synthetic peptide solution (375 ⁇ M in 50 mM Tris-HCl Buffer) 24 ⁇ l and NP solution (2 ⁇ g / ml), ColG solution (9 mg / ml) or ColH solution (4 mg / ml) 6 ⁇ l are mixed, and 30 ° C. For 30 minutes. Formic acid was added to stop the reaction. The reaction solution was loaded on reverse phase high performance liquid chromatography, and the peptide fragments produced under the following conditions were separated. Peptide fragments were detected and analyzed using Chromato-Integrator model D-2500 (Hitachi High-Technologies Co.) and Chromato-Pro software (Run Time Instruments Co.).
  • the molecular weight of the separated peptide fragment was determined using ESI-MS (equire 6000, Bruker Daltonics). Column: TSKgel ODS-120T ( ⁇ 4.6 ⁇ 250 mm, Tosho Co.) Solvent: acetonitrile with 0.1% formic acid (0-10 min: 4%, 10-40 min: 4-80%, 40-50 min: 80%), flow rate 0.5 ml / min
  • FIG. 1 and FIG. ColG and ColH did not cleave any synthetic peptide.
  • any synthetic peptide was cleaved by NP between the second amino acid (Gly) and the third amino acid (Phe) (Gly-Phe).
  • the results of FIGS. 1 and 2 are collectively shown in “Table 1”. From these results, it was confirmed that “Tyr-Gly-Phe-Tyr-Glu / Arg” can be a substrate peptide specific for NP.
  • N- [3- (2-Furyl) acryloyl] -glycine-phenylalanine-histidine amide (FA-Gly-Phe-His-NH 2 ).
  • N- [3- (2-Furyl) acryloyl] -glycine-phenylalanine-serine amide (FA-Gly-Phe-Ser-NH 2 ).
  • N- [3- (2-Furyl) acryloyl] -glycine-phenylalanine-glycine amide FA-Gly-Phe-Gly-NH 2 ).
  • N- [3- (2-Furyl) acroyl] -glycine-leucine-tyrosine amide (FA-Gly-Leu-Tyr-NH 2 ).
  • N- [3- (2-Furyl) acryloyl] -glycine-leucine-histidine amide (FA-Gly-Leu-His-NH 2 ).
  • N- [3- (2-furyl) acryloyl] -glycine-leucine-serine amide (FA-Gly-Leu-Ser-NH 2 ).
  • N- [3- (2-furyl) acryloyl] -glycine-leucine-glycine amide (FA-Gly-Leu-Gly-NH 2 ).
  • N- [3- (2-furyl) acryloyl] -glycine-isoleucine-tyrosine amide (FA-Gly-Ile-Tyr-NH 2 ).
  • N- [3- (2-Furyl) acryloyl] -glycine-isoleucine-histidine amide (FA-Gly-Ile-His-NH 2 ).
  • N- [3- (2-furyl) acryloyl] -glycine-isoleucine-serine amide (FA-Gly-Ile-Ser-NH 2 ).
  • N- [3- (2-furyl) acryloyl] -glycine-isoleucine-glycine amide (FA-Gly-Ile-Gly-NH 2 ).
  • N- [3- (2-Furyl) acryloyl] -glycine-tryptophan-tyrosine amide (FA-Gly-Trp-Tyr-NH 2 ).
  • N- [3- (2-furyl) acroyl] -glycine-tryptophan-histidine amide (FA-Gly-Trp-His-NH 2 ).
  • N- [3- (2-Furyl) acryloyl] -glycine-tryptophan-serine amide (FA-Gly-Trp-Ser-NH 2 ).
  • N- [3- (2-Furyl) acryloyl] -glycine-tryptophan-glycine amide (FA-Gly-Trp-Gly-NH 2 ).
  • NP neuropeptide
  • the specificity of NP for each synthetic peptide was analyzed by the following method. 480 ⁇ l of a synthetic peptide solution (0.4 ⁇ M, Tris-HCl buffer, pH 7.5) was pre-warmed at 37 ° C. for 5 minutes. 20 ⁇ l of NP solution, ColG solution or ColH solution was added to start the enzyme reaction. The absorbance change (344 nm) at 37 ° C. was measured using an absorption spectrum meter (Hitachi, U-3010 UV-visible scanning spectrophotometer), and the amount of degradation of each synthetic peptide was measured using the decrease in absorbance as an index. The results are shown in “Table 2”.
  • NP neutral protease
  • a region encoding NP not containing a signal peptide was amplified from the genome of Clostridium histolyticus by PCR.
  • the primer sequences are shown below.
  • Forward primer NP 5'-CGCCTAATGAGCGGGCTTTTTTCAC-3 '(SEQ ID NO: 3)
  • Reverse primer NP 5'-TACGGCTGATGTTTTTGTAATCGGCAAAC-3 '(SEQ ID NO: 4)
  • the PCR amplification product was inserted into a plasmid (pHT43, MoBiTec, Germany) to obtain an expression vector (pHT43-NP).
  • Bacillus subtilis KN2 (see Agric. Biol. Chem., 1990, 54 (5), 1307-1309) was transformed with an expression vector, and transformants were selected on a LB agar medium containing chloramphenicol.
  • the transformant was inoculated into 150 ml of medium A (composition shown below) and cultured at 30 ° C. for 18 hours.
  • Medium A composition 2% Polypepton N 1% yeast extract 1% NaCl, 20 ⁇ g / ml chloramphenicol pH 7.5
  • the culture was transferred to 9 times the amount of medium B (composition is shown below) and cultured aerobically at 37 ° C. for 3 hours. IPTG (final concentration 2 mM) and calcium acetate (100 mM) were added, and the mixture was further aerobically cultured at 37 ° C. for 24 hours.
  • Medium B composition 4% Polypepton N 2% yeast extract 2% NaCl 10 mg / ml chloramphenicol pH 7.5
  • the culture was centrifuged to obtain a culture supernatant.
  • Ammonium sulfate was added to the culture supernatant to 30% saturation and stirred at 4 ° C. for 1 hour.
  • Ammonium sulfate was added to 80% saturation to the supernatant after centrifugation and stirred at 4 ° C. for 18 hours.
  • the precipitate was collected by centrifugation and dissolved in buffer A (10 mM Tris-HCl, 1 mM calcium chloride, pH 8.0) containing ammonium sulfate at a 30% saturation concentration.
  • the supernatant was packed in a column (Toyopearl (registered trademark) Butyl-650M column, f3 x 8 cm; Tosoh Co.) equilibrated with buffer A containing ammonium sulfate at a 30% saturation concentration.
  • the column was washed with the same buffer, and then washed with buffer A containing 30% to 0% liner gradient ammonium sulfate.
  • the enzyme was eluted with buffer A containing 0% to 12.5% liner gradient ethanol. Fractions containing the enzyme were collected and dialyzed against buffer A.
  • a column (Toyopearl (registered trademark) DEAE-650S column, f2 x 5 cm; Tosoh Co.) equilibrated with buffer A was packed with the enzyme solution.
  • the column was washed with buffer A containing 10% ethanol and 50 mM sodium acetate, and then the enzyme was eluted with buffer A containing 50 mM to 200 mM liner gradient sodium acetate and 10% ethanol.
  • the fraction containing the enzyme was collected and dialyzed against the same buffer to obtain a purified enzyme.
  • SEQ ID NO: 1 substrate peptide
  • SEQ ID NO: 2 substrate peptide
  • SEQ ID NO: 3 forward primer
  • Sequence number 4 Reverse primer NP

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Abstract

L'invention concerne un substrat peptidique pour protéase comprenant la séquence d'acides aminés Gly-Xaa-Yaa (où Xaa est un acide aminé quelconque choisi parmi Phe, Leu, Ile et Trp, et Yaa est un acide aminé quelconque choisi parmi Tyr, His, Ser et Gly) en tant que substrat qui est spécifiquement coupé par une protéase neutre produite par Clostridium histolyticum.
PCT/JP2015/004901 2014-09-29 2015-09-28 Substrat peptidique pour protéase et procédé de mesure de l'activité protéase WO2016051752A1 (fr)

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JPH10500845A (ja) * 1994-05-04 1998-01-27 ノボ ノルディスク バイオテック，インコーポレイティド 増加された活性を有する新規なメタロプロテアーゼ
JP2001518791A (ja) * 1997-04-10 2001-10-16 ギルヴァーグ，チャールズ 生物学上の流体中のプロカルボキシペプチダーゼａおよびカルボキシペプチダーゼａレベルの検出方法

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