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WO1997008194A1 - Nouveau substrat fluorescent permettant d'analyser l'activite de serine protease ns3 du virus de l'hepatite c - Google Patents

Nouveau substrat fluorescent permettant d'analyser l'activite de serine protease ns3 du virus de l'hepatite c Download PDF

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Publication number
WO1997008194A1
WO1997008194A1 PCT/JP1996/002343 JP9602343W WO9708194A1 WO 1997008194 A1 WO1997008194 A1 WO 1997008194A1 JP 9602343 W JP9602343 W JP 9602343W WO 9708194 A1 WO9708194 A1 WO 9708194A1
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Prior art keywords
protease
substrate
sequence
lys
seq
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PCT/JP1996/002343
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English (en)
Japanese (ja)
Inventor
Kayo Yamaji
Yasuaki Shimizu
Yasuhiko Masuho
Kunitada Shimotohno
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Rational Drug Design Laboratories
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Priority to AU67541/96A priority Critical patent/AU6754196A/en
Publication of WO1997008194A1 publication Critical patent/WO1997008194A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • 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
    • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
    • C12Q1/18—Testing for antimicrobial activity of a material
    • 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
    • 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
    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/24011—Flaviviridae
    • C12N2770/24211—Hepacivirus, e.g. hepatitis C virus, hepatitis G virus
    • C12N2770/24222—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/005—Assays involving biological materials from specific organisms or of a specific nature from viruses
    • G01N2333/08—RNA viruses
    • G01N2333/18—Togaviridae; Flaviviridae

Definitions

  • the present invention relates to a novel modified peptide for measuring the NS3 protease activity of HCV in the presence of a hepatitis C virus (hereinafter sometimes abbreviated as “HCV”) NS4A peptide, the modified peptide and the NS4A And a method for measuring the activity of the NS3 protease in the presence of the NS4A-derived peptide using the modified peptide.
  • HCV hepatitis C virus
  • Hepatitis C virus is the causative virus of hepatitis C. It is said that hepatitis C has a large number of patients, tends to become chronic, and has a high probability of progressing to cirrhosis and liver cancer (HJ Alter et al., N. Engl. J. Med. 321, 1494-1500 (1 989) Natl. Acad. Sci. USA 87, 6547-6549 (1990): K. Shimotohno, Semin. Virol. 4, 305-312 (1993)] This is the problem above. Therefore, the drug can be said to be the most sought-after drug for viral diseases along with AIDS drug. At present, interferon is used for the treatment of hepatitis C, but its efficacy is low and its therapeutic effect is said to be limited.
  • the HCV genome encodes a single-stranded RNA (+ strand) consisting of 9400 bases, and encodes a single polyprotein of about 3000 amino acids.
  • This precursor protein contains nine types of viral proteins in order from the N-terminus (NH-C-El-E2-NS2-NS3-NS4A-NS4B-NS5A-NS5B- (C00H) [MJ Selby et al. , J. Gen. Virol., 74, 1103-1113 (1993): A. Grakoui et al., J. Virol., 67, 1385-1395 (1993): L. To mei et al., J. Virol., 67, 4017-4026 (1993)] .
  • Polyproteins are processed by a host cell-derived protease and two proteases encoded by the virus itself (NS3 protease and cprol), providing the proteins necessary for virus growth.
  • NS3 protease activity is present in the N-terminal one-third of non-structural protein 3 (NS3), and the four sites within the non-structural region that encode proteins required for viral replication (each cleavage site) The site is called “NS3 / 4A”, “NS4A / 4B”, “NS4B / 5A”, “NS5A / 5B”) [AC Grakoui et al., J. Virol. 67, 2832-2843 (1993)].
  • NS3 protease Of the four sites that are cleaved by NS3 protease, the three sites that are cleaved by trans (NS4A / 4B, NS4B / 5A, NS5A / 5B) are cysteine at the P1 position. It has an unknown substrate specificity. As described above, NS3 protease is necessary for virus growth and has a different substrate specificity from host protease, making it one of the potential targets for anti-HCV drugs. Is considered one. That is, it is considered that by screening for an NS3 protease inhibitor, a strong candidate for an anti-HCV drug can be found.
  • NS3 protease activity has been determined by co-expressing proteases and substrates in an in vitro transcription-translation system or intracellular expression system and confirming substrate cleavage by immunoprecipitation or Western blot.
  • Synthetic substrates utilizing intramolecular fluorescence quenching are already known, and have a quencher and a fluorophore across the cleavage point of the substrate sequence, and the fluorescence is suppressed by the quencher before cleavage by the enzyme. Thereafter, the quenching is released and the fluorescence intensity is increased.
  • This substrate includes stromlysin 1 (matrix proteinase-3) CH.
  • the present invention is to develop a fast, simple, highly sensitive and multi-processable cascade system of NS3 protease necessary for screening of NS3 protease inhibitors, and in particular, a novel synthetic substrate used in the cascade system.
  • the task is to provide Disclosure of the invention
  • the present inventors have conducted research mainly on the search for a suitable synthetic substrate for the purpose of developing an efficient NS3 protease protease system.
  • the present inventors initially attempted to construct an Atsushi system using a synthetic substrate using intramolecular fluorescence quenching by adding NS3 protease alone, but NS3 protease itself cleaves the substrate. Due to the low activity, it was not possible to construct a practical and efficient assay system. That is, it was not possible to establish an Atsey system for screening NS3 protease inhibitors easily and in large amounts. Thus, the present inventors have further studied and as a result, have paid attention to constructing an assay system in the presence of NS4A.
  • the present inventors have developed an atsey system utilizing a synthetic substrate utilizing intramolecular fluorescence quenching which is highly sensitive and simple and is very practical because of the HCV protease which has been desired to be realized. For the first time, it can be used for activity measurement. That is, the present inventors have made a very large contribution to the industry in connection with establishing the first practical and easy protease-based protease system for hepatitis C virus that can be practically used.
  • the present inventors have searched for a substrate utilizing intramolecular fluorescence quenching suitable for this system.
  • Substrates utilizing intramolecular fluorescence quenching can be obtained by inserting an enzyme recognition sequence between the quencher and the fluorophore.
  • the spatial distance between the quencher and the fluorophore is too large, the quenching effect of the quencher will not be obtained, and the background before cleavage will be high, making it unsuitable as a substrate for activity measurement.
  • the present inventors believe that one of the recognition sites for NS3 protease is the minimum unit required for cleavage based on the basic sequence (GEAGDDIVPCSMSYTWTGAL) used by N. Kakiuchi et al. For the NS3 protease protease system by HPLC. I asked.
  • PI and PI 'positions (Pl, P2, ⁇ 3 ⁇ ⁇ ⁇ from the cleavage point to the N-terminus and the C-terminus ⁇ ⁇ ⁇ ⁇ ' , ⁇ 2 ', ⁇ 3' ⁇ ⁇ ⁇ ) have been reported to be important for cleavage, but no substrate specificity studies using synthetic peptides at the in-vitro mouth have been made.
  • the synthetic peptide is used as a base, the present inventors assume that the sequence from the cleavage point to the ⁇ terminal side is from the ⁇ 4 position to isoleucine (IVPCSMS (YKDK) is preferred.
  • the C-terminal side from the cleavage point has a sequence (SMS) from P3 ′ to serine, but it is more preferable to have a sequence from P4 ′ to tyrosine (SMSY). It was found to be preferable (Example 1 described later). Therefore, it was predicted that a suitable peptide sequence could be obtained by combining the N-terminal sequence and the C-terminal sequence described above.Unexpectedly, however, the peptide sequence from P4 to P4 ', which is considered necessary for cleavage, was expected. Was found to be significantly less susceptible to enzymatic digestion (Example 1, Table 1.15).
  • the synthetic substrate for proteases has good solubility in water or biological test solutions.
  • a hydrophilic substrate is poor in the vicinity of the NS3 protease recognition site, and a synthetic substrate containing an amino acid sequence near the NS3 protease recognition site is poorly soluble. This was an obstacle to development.
  • the present inventors have conducted intensive experiments and found that it is possible to maintain water solubility while maintaining specificity, particularly by arranging Lys at the P5 position.
  • a modified peptide for measuring the activity of hepatitis C virus NS3 protease in the presence of a peptide derived from hepatitis C virus NS4A, and a fluorophore and quenching at any of the functional groups in the molecule A modified peptide having the following amino acid sequence (I) between the residues to which the fluorophore and the quencher are respectively bound (including the bound residues), N-terminal X-Asp-Lys-Ile-Val-Pro-Cys-Ser-Met-Ser-Y-Lys C-terminal (SEQ ID NO: 1)
  • a kit for measuring hepatitis C virus NS3 protease activity comprising a hepatitis C virus NS4A-derived peptide and the modified peptide according to (1);
  • a method for measuring hepatitis C virus NS3 protease activity which comprises observing cleavage of the modified peptide of (1) in the presence of a hepatitis C virus NS4A-derived peptide. Since hepatitis C virus has a subtype, a modification having a sequence obtained by adding a mutation such as substitution, deletion, or insertion to the amino acid sequence of (I) based on the amino acid sequence of the subtype.
  • the peptides are also included in the modified peptides of the present invention.
  • NS3 protease is used as a substrate not only at the NS5A / 5B junction but also at the NS4A / 4B and NS4B / 5A junctions, these amino acids are used.
  • Modified peptides obtained by the same method as the present invention based on the above are also included in the present invention.
  • the synthetic peptide substrate of the present invention is obtained by sequentially adding an amino acid to a lysyl group having a dinitrophenyl group as a quencher by referring to the method of H. Nagase et al. [JBC, 269, 20952-20957 (1994)]. It can be synthesized by a method of coupling according to the “method” or the “Boc method” [Nobuo Izumiya, et al., Basics and experiments on peptide synthesis (1985)].
  • amino acids requiring side chain protection are Fmoc-Lys (Boc) -OH, Fraoc-Asp (OtBu) -OH, Fmoc-Cys (Trt) -OH, Fmoc- Ser (tBu ) -OH, Fmoc-Tyr (tBu) -OH, Fmoc-Glu (OtBu) -OH or the like is used.
  • a combination of HOBt, NMM or H0Bt, DIEA, NMM can be used as an activator for amino acids.
  • the coupling reaction is performed by adding the activator shown above to a mixture of amino acids in advance with PyBOP / BOP or TBTU, respectively.
  • MF dimethylacetamide
  • N -Methylpyrrolidone is an example.
  • the fluorophore of the substrate in the present invention is a group having a fluorescent property, specifically, M0CA c ((7-methoxycouiar in-4-yl) acetyl) ⁇ EMNS (5-[(2-aminoethyl) amino) naphthalene-1 sulfonic acid), Abz (2-aminobenzoyl group) ⁇ or a group having an equivalent effect.
  • M0CA c ((7-methoxycouiar in-4-yl) acetyl) ⁇
  • EMNS 5-[(2-aminoethyl) amino) naphthalene-1 sulfonic acid), Abz (2-aminobenzoyl group) ⁇ or a group having an equivalent effect.
  • MOCAc MOCAc.
  • fluorescence means that molecules and atoms emit light by absorbing light (Biochemical Dictionary, 2nd edition (1992)).
  • the quencher is a group having the property of fluorescence quenching (Biochemical Dictionary, 2nd edition (1992)), specifically, ⁇ Drip (2, 4-dinitrophenyU) ⁇ DABCYL (4- (4-dimethylaminophenylazo benzoic acid) , Nba (4-nitrobenzylamide), etc., and groups having the same effect, preferably Dnp
  • the combination of a fluorophore and a quencher in the present invention is preferably MOCAc and Drip.
  • the functional group in the molecule is a primary amino group, a carboxyl group, or a hydroxyl group present in the modified peptide, in addition to the carboxyl group and the amino group at the C-terminal and the N-terminal of the modified peptide of the present invention.
  • Specific examples include an amino group of a lysine residue, a hydroxyl group of a serine residue or a tyrosine residue, and a hydroxyl group of an aspartic acid residue.
  • the activity of NS3 protease can be easily measured from the fluorescence intensity that increases with cleavage of the substrate, and no complicated operation such as HPLC is required.
  • the digestion reaction on a 96-well plate and the subsequent measurement of the fluorescence intensity are possible, so that a large amount of sample can be measured quickly.
  • the enzyme concentration and the substrate concentration are 80 / ig / ml and 86 / M, respectively, respectively.
  • the final concentrations of 8 g / ml and 250 nM can be measured sufficiently, and it can be said that the sensitivity is higher than that of the conventional Atsushi system.
  • NS4A in the present invention refers to a fragment, a non-structural protein 4A (NS4A) obtained as a result of digestion of the non-structural protein of HCV virus with NS3 protease as described above, and has a full-length having a hydrophilic region and a hydrophobic region. It means a protein of 54 amino acids.
  • the NS4A sequence to be added to the Atsushi system of the present invention is not limited to “4A18-40”, and any fragment may be used as long as it is an NS4A-derived fragment including the 22nd to 34th positions from the N-terminal.
  • 4A21-40, 4A18-37, 4A18-34, 4A21-34, and 4A22-34 are examples.
  • the number after 4A indicates the amino acid number at the N-terminal and C-terminal of each NS4A fragment counted from the N-terminal of NS4A.
  • FIG. 1 is a diagram showing mass spectrometry for equation (1).
  • FIG. 2 is a diagram showing mass spectrometry for equation (2).
  • FIG. 3 is a diagram showing mass spectrometry for equation (3).
  • FIG. 4 is a diagram showing a spectrum of fluorescence and excitation wavelength of a digestion product of NS3 protease of formula (2).
  • FIG. 5 is a graph showing the time-dependent change in the fluorescence intensity when the concentration of NS3 protease is changed when the formula 2 is used as a substrate.
  • FIG. 6 is a diagram showing the change over time in the fluorescence intensity in the presence and absence of 4A18-40 when using formula II as a substrate.
  • the following peptide substrates were added at 2.5 ⁇ 1 (final concentration: 100 M), respectively, and digestion was performed at 25 ° C for 6 hours.
  • the reaction was stopped by adding 1 // 1 of 5 M acetic acid.
  • the reaction solution after the termination of the reaction was separated by reversed-phase HPLC, and the digestibility of the substrate was determined from the decrease rate relative to the peak area of the substrate when the enzyme was not digested. The results are shown in the item of dNS4A- in Table 1 below. In the absence of 4A18-40, the peptide substrate was not sufficiently cleaved.
  • Example 1 Efficacy of HCV NS3 Protease Enzyme Reaction in the Presence of 4A18-40 and Identification of the Minimum Unit of Base Required for Cleavage by cNS3 Protease The effectiveness of HCV NS3 protease enzyme reaction in the presence of 4A18-40 was examined. Enzyme reaction solution (50 mM TrisHC1 (pH 7.6), 30 mM NaCl, 2 mM DTT) in MBP-NS3 (final concentration 2.2 M), 4A18-40 (final concentration 4.4 ⁇ M, twice the enzyme concentration) (Molar amount) to give 47.5 ⁇ 1.
  • Enzyme reaction solution 50 mM TrisHC1 (pH 7.6), 30 mM NaCl, 2 mM DTT
  • MBP-NS3 final concentration 2.2 M
  • 4A18-40 final concentration 4.4 ⁇ M, twice the enzyme concentration
  • Example 1 the enzymatic reaction time was shorter in the presence of 4A18-40 than in the absence of 4A18-40. Made it possible.
  • Example 1 the presence of 4A18-40 made it possible to measure HCV NS3 protease activity even with a short-chain peptide (substrate).
  • the substrate sequence has amino acid residues from the cleavage point of the protease to the N-terminus up to position P6, and the amino acid residues from the cleavage point to the position P3 ′. It has been found that they preferably have amino acid residues, and more preferably have amino acid residues from P6 to P4 '.
  • VPC-SMSYkd (SEQ ID NO: 14) 2/0
  • IVPC-SMSY (SEQ ID NO: 16) 3/0
  • Example 21 Inhibition of NS3 Protease Activity Using a Substrate Using Intramolecular Light Quenching
  • Dnp Preparation of Fmoc-Lys
  • Fmoc-DMPAMP resin (Nono BioChem) 2. Og (0.86 mmol) was allowed to act on 20 ml of piperidine-DMF (1: 1) for 30 minutes to perform deprotection, followed by washing with DMF three times. Fmoc-Lys (Dnp) (0.2 g; 3.5 ramol) and HOBt (0.536 g; 3.5 mraol) were dissolved in 20 ml of DCM-DMF (1: 1), and this solution was added to the resin. Further, DIPCDI (0.548 ml; 3.5 bandol) was added, and the reaction was performed for 4.5 hours. The product, Fmoc-Lys (Dnp) -DMPAMP, was washed with DMF and DCM, and dried under reduced pressure.
  • Fmoc-Lys (Dnp) -DMPAMP 35mg as a starting material (carrier), using the multiple peptide synthesizer (PSSM-8; Shimadzu), Fmoc method, standard cycle [PSSM-8 system instruction manual; Shimadzu]
  • PSSM-8 multiple peptide synthesizer
  • Fmoc method standard cycle [PSSM-8 system instruction manual; Shimadzu]
  • the peptide chain was extended.
  • Tyr, Ser, Met, Ser, Cys, Pro, Val, lie, Lys, and Asp were added in order
  • Lys was further added.
  • Ser, Met, Ser, Cys, Pro, Val, lie, Lys, and Asp were added in order.
  • Equations (1), (2), and (3) were consistent with the theoretical molecular weights ( Figures 1, 2, and 3).
  • the amino acid composition of the substrate was analyzed by a picotag amino acid analysis method using a picotag workstation and a gradient system (both manufactured by Waters).
  • the excitation and fluorescence wavelengths at which the digest of the substrate of the present invention showed the maximum fluorescence intensity were examined.
  • the excitation and emission wavelengths at which the MOCAc group gives the maximum fluorescence intensity are 328 nm and 393 nm, respectively. Therefore, first, the excitation wavelength was fixed at 328 nm, and the fluorescence wavelength was changed from 350 to 500 nm, and the spectrum was examined. Furthermore, the excitation wavelength was changed from 250 to 350 mn while the fluorescence wavelength was fixed at 393 ⁇ .
  • the fluorescence intensity showed the maximum value when the excitation and fluorescence wavelengths were set to 318 nm and 395 nm, respectively.
  • the other peak around 29 Onm is the peak derived from the enzyme solution, and only the enzyme is added to the reaction solution without adding the substrate, and the spectrum of the fluorescence wavelength is examined at an excitation wavelength of 318 nm. And fluorescence were hardly detected, and thus did not overlap with the fluorescence spectrum of the substrate. Equations (2) and (3) showed the same result. Therefore, the excitation and emission wavelengths of 318 nm and 395 nm were used for the fluorescence measurement of the substrate of the present invention (see FIG. 4). Next, the activity of NS3 protease was measured with a 96-well plate reader using the synthetic substrate according to the present invention.
  • the fluorescent peptide substrate of the formula II showed sufficient fluorescence intensity only by enzymatic digestion in the presence of the NS4A-derived peptide fragment. Similar results were obtained for the fluorescent peptide substrates of formulas (2) and (3). Industrial applicability
  • NS3 protease By measuring the activity of NS3 protease using the substrate of the present invention in the presence of the NS4A-derived peptide, it is possible to measure the activity of NS3 protease quickly and with high selectivity. Became. In addition, since the cleavage of the substrate can be quantified by directly observing the fluorescence, extremely simple measurement without complicated operations such as HPLC has become possible. By using the present invention, screening of an NS3 protease inhibitor that can be used as an anti-HCV agent can be easily performed.
  • Xaa means a single bond or Lys.
  • Xaa means a single bond or Tyr.
  • Lys Lys Gly Asp Asp lie Val Pro Cys Ser Met Ser Tyr Thr 1 5 10
  • Lys Asp Lys lie Val Pro Cys Ser Met Ser Tyr 1 5 10

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Abstract

Cette invention concerne un substrat capable d'accroître fortement l'activité d'incision d'un substrat par une protéase NS3 du virus de l'hépatite C. Ce substrat est traité à l'aide de protéase NS3 et en présence d'un peptide provenant de NS4 d'hépatite C, ainsi que d'une séquence d'acides aminés pouvant servir de site de clivage à un substrat peptidique, ce dernier étant utilisé dans un système d'analyse de l'activité de protéase NS3 en fonction des changements observés dans l'intensité de la fluorescence. Cette invention permet d'obtenir un système d'analyse de protéase NS3 pouvant être utilisé dans la séparation d'inhibiteurs de protéase NS3, ce qui permet d'effectuer des traitements rapides, pratiques, multiples et d'une grande sensibilité.
PCT/JP1996/002343 1995-08-25 1996-08-22 Nouveau substrat fluorescent permettant d'analyser l'activite de serine protease ns3 du virus de l'hepatite c WO1997008194A1 (fr)

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AU67541/96A AU6754196A (en) 1995-08-25 1996-08-22 Novel fluorescent substrate for assaying activity of hepatitis c virus ns3 serine protease

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JP21795095 1995-08-25
JP7/217950 1995-08-25

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Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5763196A (en) * 1996-01-26 1998-06-09 Boehringer Mannheim Corporation Assays using cross-linked polypeptide fragments of β-galactosidase
US5976783A (en) * 1996-01-26 1999-11-02 Boehringer Mannheim Corporation Bis-maleimido cross-linking agents
US5976857A (en) * 1996-01-26 1999-11-02 Boehringer Mannheim Corporation Cross-linked polypeptide fragments of β-galactosidase
US6387686B2 (en) 2000-02-25 2002-05-14 Biofocus Discovery Limited Methods and compositions for identifying a polynucleotide encoding a protease
JP2010502184A (ja) * 2006-08-28 2010-01-28 バーテックス ファーマシューティカルズ インコーポレイテッド プロテアーゼ阻害剤の同定方法

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH03172196A (ja) * 1989-11-03 1991-07-25 Abbott Lab 蛍光発生基質およびそれを用いたタンパク質分解酵素の検出方法

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH03172196A (ja) * 1989-11-03 1991-07-25 Abbott Lab 蛍光発生基質およびそれを用いたタンパク質分解酵素の検出方法

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, Vol. 210, No. 3, (25.03.95), N. KAKIUCHI et al., "Bacterial Expression and Analysis of Cleavage Activity of HCV Serine Proteinase", p. 1059-1065. *
JOURNAL OF VIROLOGY, Vol. 68, No. 11, (1994), Y. KOMADA et al., "Substrate Requirements of Hepatitis C Virus Serine Proteinase for Intermolecular Polypeptide Cleavage in Escherichia Coli", p. 7351-7357. *

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5763196A (en) * 1996-01-26 1998-06-09 Boehringer Mannheim Corporation Assays using cross-linked polypeptide fragments of β-galactosidase
US5976783A (en) * 1996-01-26 1999-11-02 Boehringer Mannheim Corporation Bis-maleimido cross-linking agents
US5976857A (en) * 1996-01-26 1999-11-02 Boehringer Mannheim Corporation Cross-linked polypeptide fragments of β-galactosidase
US6387686B2 (en) 2000-02-25 2002-05-14 Biofocus Discovery Limited Methods and compositions for identifying a polynucleotide encoding a protease
JP2010502184A (ja) * 2006-08-28 2010-01-28 バーテックス ファーマシューティカルズ インコーポレイテッド プロテアーゼ阻害剤の同定方法

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