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WO2015188388A1 - 蛋白酶 - Google Patents

蛋白酶 Download PDF

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Publication number
WO2015188388A1
WO2015188388A1 PCT/CN2014/079894 CN2014079894W WO2015188388A1 WO 2015188388 A1 WO2015188388 A1 WO 2015188388A1 CN 2014079894 W CN2014079894 W CN 2014079894W WO 2015188388 A1 WO2015188388 A1 WO 2015188388A1
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WO
WIPO (PCT)
Prior art keywords
protease
ddro
domain
gene
rdrm
Prior art date
Application number
PCT/CN2014/079894
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English (en)
French (fr)
Inventor
华跃进
王云光
王梁燕
Original Assignee
浙江大学
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Filing date
Publication date
Application filed by 浙江大学 filed Critical 浙江大学
Priority to PCT/CN2014/079894 priority Critical patent/WO2015188388A1/zh
Priority to US14/737,514 priority patent/US9299330B2/en
Publication of WO2015188388A1 publication Critical patent/WO2015188388A1/zh
Priority to US15/378,035 priority patent/US10316310B2/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C12N9/52—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from bacteria or Archaea
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
    • G—PHYSICS
    • G10—MUSICAL INSTRUMENTS; ACOUSTICS
    • G10H—ELECTROPHONIC MUSICAL INSTRUMENTS; INSTRUMENTS IN WHICH THE TONES ARE GENERATED BY ELECTROMECHANICAL MEANS OR ELECTRONIC GENERATORS, OR IN WHICH THE TONES ARE SYNTHESISED FROM A DATA STORE
    • G10H1/00—Details of electrophonic musical instruments
    • G10H1/0033—Recording/reproducing or transmission of music for electrophonic musical instruments
    • G10H1/0041—Recording/reproducing or transmission of music for electrophonic musical instruments in coded form
    • G10H1/0058—Transmission between separate instruments or between individual components of a musical system
    • G10H1/0066—Transmission between separate instruments or between individual components of a musical system using a MIDI interface
    • G—PHYSICS
    • G10—MUSICAL INSTRUMENTS; ACOUSTICS
    • G10H—ELECTROPHONIC MUSICAL INSTRUMENTS; INSTRUMENTS IN WHICH THE TONES ARE GENERATED BY ELECTROMECHANICAL MEANS OR ELECTRONIC GENERATORS, OR IN WHICH THE TONES ARE SYNTHESISED FROM A DATA STORE
    • G10H1/00—Details of electrophonic musical instruments
    • G10H1/02—Means for controlling the tone frequencies, e.g. attack or decay; Means for producing special musical effects, e.g. vibratos or glissandos
    • G10H1/04—Means for controlling the tone frequencies, e.g. attack or decay; Means for producing special musical effects, e.g. vibratos or glissandos by additional modulation
    • G10H1/053—Means for controlling the tone frequencies, e.g. attack or decay; Means for producing special musical effects, e.g. vibratos or glissandos by additional modulation during execution only
    • G10H1/055—Means for controlling the tone frequencies, e.g. attack or decay; Means for producing special musical effects, e.g. vibratos or glissandos by additional modulation during execution only by switches with variable impedance elements
    • G10H1/0551—Means for controlling the tone frequencies, e.g. attack or decay; Means for producing special musical effects, e.g. vibratos or glissandos by additional modulation during execution only by switches with variable impedance elements using variable capacitors
    • G—PHYSICS
    • G10—MUSICAL INSTRUMENTS; ACOUSTICS
    • G10H—ELECTROPHONIC MUSICAL INSTRUMENTS; INSTRUMENTS IN WHICH THE TONES ARE GENERATED BY ELECTROMECHANICAL MEANS OR ELECTRONIC GENERATORS, OR IN WHICH THE TONES ARE SYNTHESISED FROM A DATA STORE
    • G10H1/00—Details of electrophonic musical instruments
    • G10H1/18—Selecting circuits
    • G—PHYSICS
    • G10—MUSICAL INSTRUMENTS; ACOUSTICS
    • G10H—ELECTROPHONIC MUSICAL INSTRUMENTS; INSTRUMENTS IN WHICH THE TONES ARE GENERATED BY ELECTROMECHANICAL MEANS OR ELECTRONIC GENERATORS, OR IN WHICH THE TONES ARE SYNTHESISED FROM A DATA STORE
    • G10H2220/00—Input/output interfacing specifically adapted for electrophonic musical tools or instruments
    • G10H2220/091—Graphical user interface [GUI] specifically adapted for electrophonic musical instruments, e.g. interactive musical displays, musical instrument icons or menus; Details of user interactions therewith
    • G10H2220/096—Graphical user interface [GUI] specifically adapted for electrophonic musical instruments, e.g. interactive musical displays, musical instrument icons or menus; Details of user interactions therewith using a touch screen
    • G—PHYSICS
    • G10—MUSICAL INSTRUMENTS; ACOUSTICS
    • G10H—ELECTROPHONIC MUSICAL INSTRUMENTS; INSTRUMENTS IN WHICH THE TONES ARE GENERATED BY ELECTROMECHANICAL MEANS OR ELECTRONIC GENERATORS, OR IN WHICH THE TONES ARE SYNTHESISED FROM A DATA STORE
    • G10H2220/00—Input/output interfacing specifically adapted for electrophonic musical tools or instruments
    • G10H2220/091—Graphical user interface [GUI] specifically adapted for electrophonic musical instruments, e.g. interactive musical displays, musical instrument icons or menus; Details of user interactions therewith
    • G10H2220/101—Graphical user interface [GUI] specifically adapted for electrophonic musical instruments, e.g. interactive musical displays, musical instrument icons or menus; Details of user interactions therewith for graphical creation, edition or control of musical data or parameters
    • G10H2220/121—Graphical user interface [GUI] specifically adapted for electrophonic musical instruments, e.g. interactive musical displays, musical instrument icons or menus; Details of user interactions therewith for graphical creation, edition or control of musical data or parameters for graphical editing of a musical score, staff or tablature
    • G—PHYSICS
    • G10—MUSICAL INSTRUMENTS; ACOUSTICS
    • G10H—ELECTROPHONIC MUSICAL INSTRUMENTS; INSTRUMENTS IN WHICH THE TONES ARE GENERATED BY ELECTROMECHANICAL MEANS OR ELECTRONIC GENERATORS, OR IN WHICH THE TONES ARE SYNTHESISED FROM A DATA STORE
    • G10H2220/00—Input/output interfacing specifically adapted for electrophonic musical tools or instruments
    • G10H2220/155—User input interfaces for electrophonic musical instruments
    • G10H2220/221—Keyboards, i.e. configuration of several keys or key-like input devices relative to one another
    • G10H2220/241—Keyboards, i.e. configuration of several keys or key-like input devices relative to one another on touchscreens, i.e. keys, frets, strings, tablature or staff displayed on a touchscreen display for note input purposes
    • G—PHYSICS
    • G10—MUSICAL INSTRUMENTS; ACOUSTICS
    • G10H—ELECTROPHONIC MUSICAL INSTRUMENTS; INSTRUMENTS IN WHICH THE TONES ARE GENERATED BY ELECTROMECHANICAL MEANS OR ELECTRONIC GENERATORS, OR IN WHICH THE TONES ARE SYNTHESISED FROM A DATA STORE
    • G10H2220/00—Input/output interfacing specifically adapted for electrophonic musical tools or instruments
    • G10H2220/155—User input interfaces for electrophonic musical instruments
    • G10H2220/265—Key design details; Special characteristics of individual keys of a keyboard; Key-like musical input devices, e.g. finger sensors, pedals, potentiometers, selectors
    • G10H2220/275—Switching mechanism or sensor details of individual keys, e.g. details of key contacts, hall effect or piezoelectric sensors used for key position or movement sensing purposes; Mounting thereof
    • G—PHYSICS
    • G10—MUSICAL INSTRUMENTS; ACOUSTICS
    • G10H—ELECTROPHONIC MUSICAL INSTRUMENTS; INSTRUMENTS IN WHICH THE TONES ARE GENERATED BY ELECTROMECHANICAL MEANS OR ELECTRONIC GENERATORS, OR IN WHICH THE TONES ARE SYNTHESISED FROM A DATA STORE
    • G10H2220/00—Input/output interfacing specifically adapted for electrophonic musical tools or instruments
    • G10H2220/461—Transducers, i.e. details, positioning or use of assemblies to detect and convert mechanical vibrations or mechanical strains into an electrical signal, e.g. audio, trigger or control signal

Definitions

  • Proteases are a generic term for a class of enzymes that hydrolyze protein kinetic bonds and catalyze the hydrolysis of peptide bonds in proteins. Proteases are widely found in animals, plants and microorganisms. So far, more than 100 commercial proteases have been on the international market. Due to limited animal and plant resources, industrially produced protease preparations are mainly derived from microorganisms, and are prepared by microbial extraction using Bacillus subtilis, yeast, mold, Escherichia coli and the like. With the deepening of protease research, its industrial application has attracted more and more people's attention.
  • proteases have been widely used in the fields of fur, leather, silk, medicine, food, brewing, and oil drilling.
  • Protease can also be used for silk degumming, meat tenderization, and wine clarification. It can be used clinically for medicinal purposes, for example for the treatment of symptoms such as indigestion, bronchitis and vasculitis.
  • the protease-added washing powder can effectively remove the stains and protein stains on the clothes.
  • proteases are widely used in biochemical molecular experiments, and as a scalpel for proteins, they are indispensable for life science research.
  • Radiation-resistant bacterium is an extremely environmental microorganism known for its extreme resistance to extreme conditions such as ionizing radiation, ultraviolet radiation, drying and oxidative stress. Part of its super-resistance is attributed to its 3 ⁇ 4o/gene (gene name dr-0167, NCBI-GenelD: 1798483). It plays an integral role in the repair of genetic damage. 3 ⁇ 4o / Gene Expression Product
  • the Pprl protein (NCBI GI: 15805204) consists of 328 amino acids and contains three functional domains, the zinc finger protease domain, the helix-helical domain, and the GAF domain. However, to date, the protease activity and enzyme substrate of Pp:rl have not been found.
  • the object of the invention is to provide a protease.
  • protease having a zinc finger protein alcohol domain, a helix angle-helix domain and a GAF domain, said protease holoenzyme and an independent structural zinc finger-protease domain having an equivalent protease activity of said zinc finger protease
  • the domain core protein sequence is set forth in SEQ ID NO: 1.
  • One of the substrates of the protease is Radiation-resistant bacterium / fei 0 cci ⁇ radiodurans, ATCC No, 13939 transcription factor DdrO (Gene ID: 1798752; NP-296294, 1), the transcription factor can be combined with the DNA damage emergency response and repair gene promoter of the RDRM-containing site in the in vitro and in vivo.
  • the substrate specific sequence of the protease is ELXGXR, wherein X is any essential amino acid and the cleavage position is between the second and third amino acid residues.
  • the protease digestion reaction buffer contains 100 200 mM NaC 10 - 50 mM Tris-HCl 8, 0, 1 mM DTT, 2. 0-5. 0 mM M [iCl 2 ] .
  • the protease has an enzymatic activity temperature in the range of 4 65 ° C, and preferably the protease has an activity temperature in the range of 35 - 40 °C.
  • the gene promoters include rfrf3 ⁇ 4?, dr0099, draOlS dr0219, dr0326, dra0346, dr()423, dr0596 dr0906 drl()39, d-rll43, drl-289, d:rl696, drl771, drl775, drl913, Drl92 d: r2256, dr2275, dr'2336., di'2574.
  • the binding reaction buffer to the gene promoter containing the RDRM site contains 100-200 raM NaC 20-50 mM Tri s-IICl 8, 0, 5- 10 raM MgCl 2 , and the reaction temperature is 30° (:.
  • the minimal sequence of the transcription factor binding is the RDRM site within the bacterium resistant to Radococcus.
  • the protease is derived from Deinococcus radiodurans.
  • the protease of the present invention has a high specificity for enzymatic cleavage, good heat resistance and high enzymatic cleavage efficiency, and provides a tool for basic research and industrial application, and opens up a road.
  • Figure 1 is a diagram showing the in vitro digestion of Pprl protease.
  • I purified DdrO protein
  • 2 purified Pprl protein
  • 3 DdrO and Pprl digestion reaction
  • 4 Fermentas pre-stained protein Marker SM0671.
  • Figure 2 shows the in vitro digestion of Pprl protease by Western blotti ng. Among them, 1 : After purification, DdrO protein was detected by Western blotting; 2: DdrO was digested with Pprl and detected by Western blotting. The two bands are the DdrO protein and the large fragment of the DdrO protein after digestion.
  • Figure 3 is a C-terminal sequencing of a large fragment of the Pdrl protease substrate after DdrO digestion.
  • peak A the molecular weight of the small fragment after DdrO digestion
  • peak B the molecular weight of the large fragment after DdrO digestion
  • peak C the molecular weight of the DdrO protein.
  • Figure 4 is a substrate-specific cleavage sequence of the Pprf protease.
  • FIG. 5 is a digestion model of Pprl protease.
  • the amino acid residue indicated in the red body is the restriction endonuclease recognition sequence of Pprl protease, wherein X represents a variable amino acid residue, which may be any essential amino acid.
  • the position indicated by the arrow is its cleavage site.
  • Figure 6 is a SDS-PAGE diagram of the activity of different metal ions on the protease Pprl enzyme.
  • control reactions without metal ions from left to right, are the control reactions without metal ions; the control reaction with EDTA; the reaction of Ca 2+ at the pressure; the reaction with Cu 2 '; the reaction with Fe 2 ; the Mg 2+ Reaction; reaction of Ni 2+ ; reaction of Mn 2+ ; reaction of Zn 2 -.
  • concentration of various metal ions and EDTA is lniM/L.
  • Figure 7 shows that DdrO binds to a gene promoter containing an RDRM site in vitro.
  • CK is the control without DdrO protein; the other is the gene promoter containing RDRM site, and ⁇ ⁇ /, ⁇ ? and ⁇ ⁇ ⁇ 3 ⁇ 43 ⁇ 4 did not appear DNA band migration, the reason It is the sequence alignment of its RDRM site that is less trustworthy.
  • Figure 8 shows that the RD site of the gene promoter is required for in vitro DdrO binding.
  • Seven gene promoters containing RDRM sites were selected as the study. Its ⁇ ⁇ / ⁇ is the full promoter of the re gene, including the RDRM site; and Rred is the _ ⁇ 4 gene promoter that does not contain the RDRM site. Other gene promoters are analogous.
  • the gene promoter containing the RDRM site binds to the DdrO protein and does not contain the RDRM site but does not bind to the DdrO protein in a concentration range of 0. ⁇ /L to 2. 4p /L.
  • Figure 9 shows the DfO binding to the DNA damage emergency response and repair gene promoter in vivo.
  • the expression amount is constant before and after treatment.
  • the number of promoters of the selected gene enriched with the specific DdrO protein rabbit antibody is three to six times more than that of the non-specific antibody.
  • Figure 10 shows the effect of DdrO protein on the transcription of DDRR gene in wild strain R1 before and after irradiation treatment.
  • Before IR refers to the sample before irradiation
  • 35inin after IR refers to the sample that resumes culture for 35 minutes after irradiation
  • 90mi n after IR refers to the sample that has been restored to culture for 90 minutes after irradiation;
  • Figure 1 1 shows the effect of Dd) protein on the transcription of DDRR gene in the '73' strain YR1 before and after irradiation treatment.
  • IR Before IR, refers to the sample before irradiation; 35min after IR, refers to the sample that resumes culture for 35 minutes after irradiation; 90mi n after IR, refers to the sample that has been cultured for 90 minutes after irradiation.
  • the strain used in the examples of the present invention is Deinococais radiodurans (ATCC No. 13939), and the Escherichia coli expression strain BL21 (DE3) Chemical ly Competent Cel l (genotype: F- om l hs(E ⁇ (rB mB- ) gal dcm (DE3) ), E.
  • the purified Pprl protein was reacted with its substrate protein DdrO in reaction buffer (150 mM NaCl, 20 mM Tris-HCl 8.0, 1 mM DTT, 2. OmM MnCl 2 ) for 40 minutes.
  • the Pprl protein can be used to cut the DdrO enzyme into two segments by SDS PAGE and mass spectrometry.
  • the specific sequences of the Pprl protease cleavage substrate were ELRGKR, ELRGAR, ELRGER, ELAGKR, ELAGAR and ELAGER by point mutation of the amino acid residue near the DdrO restriction site.
  • the cleavage position can be obtained between the second and third amino acid residues (Fig. 1, Fig. 2, Fig. 3, Fig. 4, Fig. 5).
  • the optimum temperature for the pprf protease to digest with the substrate is between 35 and 40 ⁇ . Protease activity is highest in this temperature range. When the temperature is between 50 and 55, the protease activity still exists, which is about one-third lower. At a temperature of 65 ⁇ , the activity is weak. Therefore, Pprl protease has a wide temperature range and is a temperature-resistant protease.
  • the Pprl protein requires the presence of the metal ion Mn 2+ when exercising protease activity.
  • the activity was best at a final concentration of Mn 2+ of 2 mM/L (Fig. 6).
  • DdrO was reacted with PiirO3 ⁇ 4 ⁇ , PiiraCS and ⁇ 5 3 ⁇ 4 full promoter in binding buffer (200 mM NaCl, 50 mM Tris-HCl 8, 0, 10 mM MgCl 2 ) for 40 minutes.
  • binding buffer 200 mM NaCl, 50 mM Tris-HCl 8, 0, 10 mM MgCl 2
  • the migration of DNA bands was detected by 12% TB PAGE gel.
  • DdrO binds to the gene promoter containing the RDRM site in vivo
  • the wild strains R1 and ⁇ r/3 ⁇ 43 ⁇ 4 strain YR1 were collected by centrifugation, RNA was extracted, reversed, and then qRT PCI3 ⁇ 4t3 ⁇ 4 in vivo iirC, ch'2340, dr2574, drOOTO, dra0346, dr'0423,
  • the purified PprT protein was reacted with its substrate protein DdrO in reaction buffer (150 mM Na, Cl, 20 mM Tris-IICI 8, 0, 1 mM DTT, 3, OmM MnCl 2 ) for 40 minutes.
  • the Pprf protein can be used to cut the DdrO enzyme into two segments by SDS PAGE and mass spectrometry.
  • the specific sequences of the Pprl protease cleavage substrate were ELRGKR, ELRGAR, ELAGKR and ELAGAR by point mutation of the DdrO cleavage site near the amino acid residue.
  • the C-terminal mass spectrum of the protein can be used to obtain a cleavage position between the second and third amino acid residues (Fig. 1, Fig. 2, Fig. 3, Fig. 4, Fig. 5).
  • the optimum temperature for Pprl protease to digest with the substrate is between 35-40 °C. Protease activity is highest in this temperature range, and ⁇ remains. At 4 ⁇ , it also showed weaker protease activity. At a temperature of 65 °C, the activity is weak. Therefore, Pprl protease has a wide temperature range and is a temperature-resistant proteinase.
  • the Pprf protein requires the presence of the metal ion Mn 2 ' when exercising protease activity.
  • Mn 2 ' When the final concentration of Mn 2 ' is 2 mM/L, the activity is preferred.
  • Other divalent ions such as Ni 2+ , Zn 2+ , etc., have a final concentration greater than 0.25 mM/L, which inhibits protease activity (Fig. 6).
  • the wild strain R1 is compared with the 3 ⁇ 4ir_ mutant YR1 intracellular D damage emergency response and the repair gene transcription level is up-regulated
  • wild strain R1 and ⁇ r/3 ⁇ 4 strain YR1 were cultured in the medium for 35 minutes, the cells were collected by centrifugation, RNA was extracted, reverse transcription was performed, and dRT was detected by qRT PCR. , di'2340, d'2574, di'0070, dra() 346 d: r0423, £ir'i?ft3 ⁇ 4 ⁇ ⁇ ⁇ transcription level of the gene. Among them, ' ⁇ was used as a negative gene, and the expression level was constant before and after treatment. The results showed that in the early stage of irradiation treatment, the DNA damage emergency response and repair gene transcription level of wild strain R1 were up-regulated, while the deletion strain YR1 did not undergo transcriptional changes in vivo (Fig. 10, Fig. 11).
  • the purified Pprl protein was reacted with its substrate protein Dcl.rO in reaction buffer (150 raM NaCK 20rnM Tris-HCl 8, 0, 1 mM DTT, 5, OrnM MnCl 2 ) for 40 minutes.
  • the Pprl protein can be used to cut the DdrO enzyme into two segments by SDS PAGE and mass spectrometry.
  • the specific sequences of the Pprl protease cleavage substrate were ELRGKR, ELRGAR, ELRGER, ELAGKR, ELAGAR and ELAGER by point mutation of the amino acid residue near the DdrO restriction site.
  • the cleavage position can be obtained between the second and third amino acid residues (Fig. 1, Fig. 2, Fig. 3, Fig. 4, Fig. 5).
  • the optimum temperature for Pprl protease to digest with the substrate is between 35 40 ⁇ . Protease activity is highest in this temperature range, and ⁇ remains. At 4 ⁇ , it also showed weaker protease activity. When the temperature is between 50-55 ° C, the protease activity still exists, about a third lower.
  • the Pprl protein requires the presence of the metal ion Mn 2+ when exercising protease activity.
  • the final concentration of Mn 2 is At 5 mM/L, activity remained.
  • Other divalent ions such as Fe 24 , Cu 2+ , etc., with a final concentration greater than 0, 25 mM/L, have an inhibitory effect on protease activity (Fig. 6).
  • DdrO binds in vivo DNA damage emergency response and repair gene promoter
  • the DNA sequence bound to the DdrO protein was purified by DdrO antibody, and the negative control gene ⁇ 03 ⁇ 4 was quantitatively detected by RT-PCR. Abundance. The results showed that the specific DdiO antibody was able to enrich the number of promoters of the selected gene with the same number of non-specific antibodies (Fig. 9).
  • the expression level is constant before and after treatment.
  • the results showed that in the middle and late stages of irradiation treatment, the DNA response levels of the DNA response and the repair gene in the wild strain R1 and the 3 ⁇ 4ar/deficient strain YR1 did not change (Fig. 10, Fig. 1 1 ).
  • the strain used in the examples of the present invention is Deinococcus radiodurans (ATCC No. 13939), but according to the teachings and teachings of the present invention, any synthetic or other naturally contained proteases and derivatives, such as having radiation resistance Sequences and similar structures and functions of the genomic peptide Pprl homologous are also within the scope of the present invention.

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Abstract

本发明提供一种蛋白酶，包括锌指-蛋白酶结构域、螺旋-转角-螺旋结构域和GAF结构域，所述锌指-蛋白酶结构域核心蛋白序列如SEQ ID NO：1所示。

Description

蛋白酶是水解蛋白质欣键的一类酶的总称， 可以催化蛋白质中肽键的水 解。 蛋白酶广泛存在于动物、 植物和微生物中。 到目前为止， 国际市场上商 品蛋白酶 100种以上。 由于动植物资源有限， 工业上生产蛋白酶制剂主要来 自微生物， 利用枯草杆菌、 酵母、 霉菌、 大肠杆菌等微生物提取制备。 随着 蛋白酶研究的深入， 它的工业应用越来越引起了人们的广泛关注。
蛋白酶己广泛应用在毛皮、 皮革、 丝绸、 医药、 食品、 酿造、 石油钻井 等领域。 利用蛋白酶， 皮革工业的脱毛软化， 既节省时间， 又改善劳动卫生 条件。 蛋白酶还可用于蚕丝脱胶、 肉类嫩化、 酒类澄清。 临床上可作药用， 例如用于消化不良、 支气管炎、 脉管炎等症状的治疗。 加入蛋白酶的洗衣粉 能高效去除衣物上的愈渍和蛋白污物。另外， 蛋白酶广泛应用于生化分子实 验， 作为蛋白质的手术刀， 是生命科学研究不可或缺的。
耐辐射奇球菌是一种极端环境微生物， 以对电离辐射、 紫外射线、 干燥和 氧化压力等极端条件表现极强抗性而著称。 它的这种超强抗性有一部分归功于 其体内 ¾o /基因 （基因名 dr— 0167, NCBI- GenelD : 1798483 )。 它是在基因损伤 修复中起整体调控作用。 ¾o /基因表达的产物 Pprl蛋白 （NCBI GI : 15805204 ) 由 328个氨基酸组成， 含 3个功能结构域， 分别是锌指 蛋白酶结构域， 螺旋 转角-螺旋结构域， GAF结构域。 但是至今为止， Pp:rl的蛋白酶活性和酶底物从 未被发现。
-本发明的目的是提供一种蛋白酶。
一种蛋白酶，具有锌指 蛋白醇结构域、 螺旋 转角--螺旋结构域和 GAF结构 域， 所述的蛋白酶全酶和独立的结构锌指-蛋白酶结构域具有同等的蛋白酶活 所述锌指 蛋白酶结构域核心蛋白序列如 SEQ ID N0 : 1所示。
所述蛋白酶的底物之一为耐辐射奇球菌/ fei 0 cci^ radiodurans, ATCC No, 13939的转录因子 DdrO (Gene ID : 1798752 ; NP— 296294, 1)， 所述的转录因 子可以在体内外结合耐辐射奇球菌内含 RDRM位点的 DNA损伤应急响应与修复基 因启动子。
所述蛋白酶的底物酶切特异性序列是 ELXGXR， 其中 X为任意一种必需氨基 酸， 切割位置在第二和第三个氨基酸残基之间。
所述蛋白酶的酶切反应缓冲液含 100 200mM NaC 10 - 50mM Tris-HCl 8, 0、 ImM DTT、 2. 0-5. 0 mM M〖iCl2。
所述的蛋白酶的酶活性温度范围为 4 65°C， 优选的所述蛋白酶的活性温度 范围为 35- 40°C。
所述的基因启动子包括 rfrf¾? »、 dr0099、 draOlS dr0219、 dr0326、 dra0346、 dr()423, dr0596 dr0906 drl()39、 d—rll43、 drl—289、 d:rl696、 drl771、 drl775、 drl913、 drl92 d:r2256、 dr2275、 dr'2336.、 di'2574。
与含 RDRM位点的基因启动子的结合反应缓沖液含 100-200 raM NaC 20-50 mM Tri s- IICl 8, 0、 5- 10 raM MgCl2，反应温度为 30° (：。
所述的转录因子结合最小序列为耐辐射奇球菌内的 RDRM位点。
所述的蛋白酶来源于耐辐射奇球菌。
本发明中蛋白酶的酶切序列特异性较高、 耐热性较好、 酶切效率较高， 为 基础研究和工业应用提供了工具， 开辟了道路。 图 1是 Pprl 蛋白酶体外酶切电泳图。
其中， I： 纯化后的 DdrO蛋白; 2： 纯化后的 Pprl蛋白； 3 : DdrO与 Pprl 酶切反应； 4: Fermentas预染蛋白 Marker SM0671 。
图 2是 Pprl 蛋白酶体外酶切 Western blotti ng 检测。 其中， 1 : 纯化后 DdrO蛋白经 Western blotting检测; 2： DdrO与 Pprl酶切反应后， 经 Western blotting检测。 两条带分别为 DdrO 蛋白和酶切后 DdrO蛋白大片段。
图 3是 Pprl 蛋白酶的底物 DdrO酶切后大片段的 C端测序。
其中， 峰 A: DdrO 酶切后， 其小片段的分子量； 峰 B: DdrO 酶切后， 其大 片段的分子量； 峰 C: DdrO 蛋白的分子量。
图 4是 Pprf 蛋白酶的底物特异性酶切序列。
其中， 从左至右， 从上至下， 分别是 Ρρι·Ι蛋白与 DdrO不同点突变蛋白的酶 切反应。 S104A， 表示 DdrO的第 104位残基 S突变成 A， 其它依次类推； WT， 表示 野生型的 DdrO蛋白。 图 5是 Pprl 蛋白酶的酶切模型。 红体标注的氨基酸残基是 Pprl 蛋白酶的酶 切识别序列， 其中 X表示可变的氨基酸残基， 可能是任意一种必需氨基酸。 箭头 所指位置是其酶切位点。
图 6是不同金属离子对蛋白酶 Pprl酶活性的 SDS- PAGE图。
其中，从左至右，分别是不加金属离子的对照反应；加 EDTA的对照反应； 力口 Ca2+的反应； 加 Cu2'的反应; 加 Fe2的反应; 力口 Mg2+的反应； 力口 Ni2+的反 应； 力口 Mn2+的反应； 力口 Zn2—的反应。 各种金属离子及 EDTA的浓度均为 lniM/L。
图 7是 DdrO在体外结合含 RDRM位点的基因启动子。
其中， 从左至右， CK为不加 DdrO蛋白的对照; 其他为含 RDRM位点的基因 启动子，而 Ρ ^^ /、Ρώ^^?与 Ρ ώ^¾¾未出现 DNA条带迁移，原因是其 RDRM 位点的序列比对信任度比较低下。
图 8是基因启动子的 RD 位点为体外 DdrO结合所必需。
选取含 RDRM位点的七个基因启动子作为研宄。 其 Φ Ρ/ ^是 re 基因的全启 动子， 包含 RDRM位点； 而 Rred则是不包含 RDRM位点的 _ ^4基因启动子。 其他 基因启动子依次类推。 含 RDRM位点的基因启动子可以与 DdrO蛋白结合， 而不包 含 RDRM位点的却不可以与 DdrO蛋白结合， 浓度范围为 0. δμΜ/L到 2. 4p /L。
图 9是 DdrO在体内能结合 DNA损伤应急响应与修复基因启动子。
其中， 因为阴性对照， 因为它是看家基因， 在处理前后表达量 恒定。用特异性的 DdrO蛋白兔抗体富集的所选基因启动子数量比非特异性抗 体多三到六倍。
图 10是在辐照处理前后野生菌株 R1体内 DdrO蛋白对 DDRR基因转录的影响。 Before IR,指輻照前的样品； 35inin after IR，指辐照后恢复培养 35分钟的 样品； 90mi n after IR, 指辐照后恢复培养 90分钟的样品；
图 1 1是在辐照处理前后 /'7¾失菌株 YR1体内 Dd )蛋白对 DDRR基因转录的 影响。
Before IR，指辐照前的样品； 35min after IR,指辐照后恢复培养 35分钟的 样品； 90mi n after IR, 指辐照后恢复培养 90分钟的样品。
以下结合附图和实施例对本发明作进一步的说明。
本发明实施例所用的菌株为耐辐射奇球菌（ Deinococais radiodurans, ATCC No. 13939 )，大肠杼菌表达菌株 BL21 (DE3) Chemical ly Competent Cel l (基因型： F- om l hs(E\ (rB mB- ) gal dcm (DE3) )，大肠杆菌克隆菌株 T:rans5 α Chemically Competent Cell (基因型: F- Φ80 iacZAMIS Δ (lacZYA~arg¥) ill 69 end l rechl AsriH 7 (rk— , !nk十) supVA-i λ - thl —1 g rk96 reiki j¾¾oA)。
^ ' (1) Pprl的蛋白酶活性和底物醇切序列特异性
将纯化的 Pprl蛋白与其底物蛋白 DdrO在反应缓冲液 (150mM NaCl、 20mM Tris-HCl 8.0, ImM DTT、 2. OmM MnCl2) 中反应 40分钟。 经 SDS PAGE电泳和质谱 分子量大小检测， Pprl蛋白可以把 DdrO酶切成两段。 通过 DdrO酶切位点附近氨 基酸残基的点突变， 获得 Pprl 蛋白酶切底物的特异性序列为 ELRGKR、 ELRGAR, ELRGER, ELAGKR, ELAGAR和 ELAGER。 另外， 经蛋白质的 C端质谱测序， 可以获得 切割位置在第二和第三个氨基酸残基之间 （图 1， 图 2， 图 3， 图 4， 图 5)。
(2) Pprl蛋白醇的酶活最适温度范围和耐温性
Pprf蛋白酶与底物发生酶切反应的最适温度在 35 40Ό之间。 在这个温度范 围内蛋白酶活性最高。 当温度在 50 55Ό之间时， 蛋白酶活性仍然存在， 约降低 了三分之一。 在温度 65Ό日寸， 活性较弱。 所以 Pprl蛋白酶活温度范围较广， 是 一种耐温蛋白酶。
(3) Pprl的蛋白酶活性依赖于金属离子 Mn2+
Pprl蛋白在行使蛋白酶活性时， 需要金属离子 Mn2+的存在。 在 Mn2+终浓度为 2mM/L时， 活性最佳 （图 6) 。
(4) DdrO在体外结合含 RDRM位点的基因启动子
在不加 DdrO情况下， 选取 c ^g因启动子在结合缓冲液 （200 mM NaCl、 50 mM Tris HCl 8, 0、 lOroM MgCl2)中反应 40分钟。 用 12% TB PAGE胶检测 DN.A条 带的迀移情况。 实验表明， 在不加 DdrO时， DNA条带未发生迁移 （图 7) 。
(5) 基因启动子的 RD 位点为体外 DdrO结合所必需
DdrO与 PiirO¾^、 PiiraCS和 Ρώ··^5 ¾等全启动子在结合缓冲液 （200 mM NaCl、 50mM Tris-HCl 8,0、 10 mM MgCl2)中反应 40分钟。 12% TB PAGE胶检测 DNA条带的迁移情况。 实验表明， 上述基因启动子均能结合 DdrO，发生条带迀移 (图 8) 。
(6) DdrO在体内结合含 RDRM位点的基因启动子
运用染色质免疫共沉淀技术， 用 DdrO抗体纯化在体内与 DdrO蛋白结合的 DNA 序列， 再由 RT PCR定量检测 Mri¾?7i7和 Ρ ί¾¾¾？等的丰度。 结果表明， 特异性的 DdrO抗体能富集所选基因启动子的数量比非特异性的抗体多三到六倍 （图 9 )
(7) 辐照处理前， 野生菌株 R1相对于 Ρ Γ/¾变株 YR1胞内 DNA损伤应急响 应与修复基因转录水平不变
在辐照处理前， 离心收集野生菌株 R1与^ r/¾¾失菌株 YR1， 提取 RNA， 反转 录， 后^ qRT PCI¾t¾体内 iirC、 ch'2340、 dr2574、 drOOTO, dra0346、 dr'0423、
^等基因的转录水平， 其中0¾¾¾¾？作为阴性基因， 基因突变前后 表达量恒定。 结果表明， 在未辐照处理前， 野生菌株 R1与^缺失菌株 YR1体内 DNA损伤应急响应与修复基因转录水平均未发生变化（图 10， 图 11)。
( 1 ) Pprl的蛋白酶活性和底物酶切序列特异性：
将纯化的 PprT蛋白与其底物蛋白 DdrO在反应缓沖液 （150mM Na,Cl、 20mM Tri s-IICI 8, 0、 ImM DTT、 3, OmM MnCl2 ) 中反应 40分钟。 经 SDS PAGE电泳和质谱 分子量大小检测， Pprf蛋白可以把 DdrO酶切成两段。 通过 DdrO酶切位点險近氨 基酸残基的点突变， 获得 Pprl 蛋白酶切底物的特异性序列为 ELRGKR、 ELRGAR、 ELAGKR和 ELAGAR。 另外， 经蛋白质的 C端质谱 ¾序， 可以获得切割位置在第二和 第三个氨基酸残基之间 （图 1， 图 2， 图 3， 图 4， 图 5 )。
( 2 ) Pprf蛋白酶的酶活最适温度范围和耐温性
Pprl蛋白酶与底物发生酶切反应的最适温度在 35-40 °C之间。 在这个温度范 围内蛋白酶活性最高， 而 ϋ一直保持着。而在 4Ό时， 也表现较弱的蛋白酶活性。 在温度 65 °C时， 活性较弱。 所以 Pprl蛋白酶活温度范围较广， 是一种耐温蛋白 酶。
(： 3 ) Pprl的蛋白酶活性依赖于金属离子 Mn2+
Pprf蛋白在行使蛋白酶活性时， 需要金属离子 Mn2'的存在。 在 Mn2'终浓度为 2mM/L时， 活性较佳。 其他二价离子， 如 Ni2+，Zn2+等， 终浓度大于 0. 25mM/L,均对 蛋白酶活性存在抑制的作用 （图 6 ) 。
( 4 ) DdrO在体外结合含 RDRM位点的基因启动子
DdrO与 rfrt¾?7i?、 dii)099、 draOlbU dr0219、 dr0326, dra0346、 dr0423、 dr()596、 ？和 ii /ft 等基因的全启动子在结合缓冲液 （100 inM NaCl、 20 rnM
Tri s-HCI 8, 0、 5 mM MgCl2)中反应 40分钟。 12% TB PAGE胶检测 DNA条带的迁 移情况。 实验表明， 上述基因启动子均能结合 DdrO，发生条带的迁移 （图 7 ) 。 (5) 基因启动子的 RD 位点为体外 DdrO结合所必需
Dd:r0与不含 RDRM位点的 Pcrt^7^ -、 ?dr0099~, Ρ ? t¾?~ tlPiirtMg^~ 基因 启动子在结合缓冲液 (100 mM NaC:l、 20 raM Tris-HCl 8,0、 5 mM MgCl2)中反应 40分钟。 用 12% ΤΒ·- PAGE胶检测 DNA条带的迀移情况。 实验表明， 上述不含 RDRM 位点的基因启动子均不能结合 DdrO,未发生条带迁移 （图 8) 。
( 6 ) DdiO在体内结合 DNA损伤应急响应与修复基因启动子
运用染色质免疫共沉淀技术， 用 Dd:r0抗体纯化在体内与 DdrO蛋白结合的 DNA 序列，
Figure imgf000007_0001
Ρ τ^Ο ^的丰度。 结果表明， 特异性的 DdrO抗体能富集所选基因启动子的数量比非特异性的抗体多三到六倍 （图 9)
(7)辐照处理后， 野生菌株 R1相对于¾ir_突变株 YR1胞内 D 损伤应急响应 与修复基因转录水平上调
经 2kGy剂量 γ射线辐照处理后， 野生菌株 R1与^ r/¾失菌株 YR1在培养基 中恢复培养 35分钟， 离心收集菌体， 提取 RNA， 反转录， 并用 qRT PCR检测体内 d:r0089、 di'2340、 d'2574、 di'0070、 dra()346 d:r0423、 £ir'i?ft¾环 Π ^基因 的转录水平。 其中 'ί^^^作为阴性基因， 处理前后表达量恒定。 结果表明， 在 辐照处理恢复前期， 野生菌株 R1体内 DNA损伤应急响应与修复基因转录水平均上 调， 而^ 缺失菌株 YR1体内未发生转录水平变化 （图 10， 图 11)。
' (l)Pprl的蛋白酶活性和底物酶切序列特异性
经纯化的 Pprl蛋白与其底物蛋白 Dcl.rO在反应缓冲液 (150raM NaCK 20rnM Tris-HCl 8,0、 ImM DTT、 5, OrnM MnCl2) 中反应 40分钟。 经 SDS PAGE电泳和质谱 分子量大小检测， Pprl蛋白可以把 DdrO酶切成两段。 通过 DdrO酶切位点附近氨 基酸残基的点突变， 获得 Pprl 蛋白酶切底物的特异性序列为 ELRGKR、 ELRGAR, ELRGER、 ELAGKR、 ELAGAR和 ELAGER。 另外， 经蛋白质的 C端质谱测序， 可以获得 切割位置在第二和第三个氨基酸残基之间 （图 1， 图 2， 图 3， 图 4， 图 5)。
(2) PprI的蛋白酶的醇活最适温度范围和耐温性
Pprl蛋白酶与底物发生酶切反应的最适温度在 35 40Ό之间。 在这个温度 范围内蛋白酶活性最高， 而 ϋ一直保持着。 而在 4Ό时， 也表现较弱的蛋白酶活 性。 当温度在 50- 55°C之间时， 蛋白酶活性仍然存在， 约降低了三分之一。
(3) Pprl的蛋白酶活性依赖于金属离子 Mn2+
Pprl蛋白在行使蛋白酶活性时， 需要金属离子 Mn2+的存在。 在 Mn2终浓度为 5mM/L时，活性仍然存在。其他二价离子，如 Fe24, Cu2+等，终浓度大于 0, 25mM/L, 均对蛋白酶活性存在抑制的作用 （图 6 ) 。
(4) DdrO在体外结合含 RD 位点的基因启动子
在不加 DdrO情况下， 选取 ?^ ¾因启动子在结合缓冲液 （200 mM NaCl、 50 mM Tr i s- IIC I 8, 0、 l OroM MgCl 2)中反应 40分钟。 用 12% TB PAGE胶检测 DN.A条 带的迁移情况。 实验表明， 在不加 DdrO时， DNA条带未发生迁移 （图 7 ) 。
(5)基因启动子的 RDRM位点是体外 DdrO结合所必需
DdrO与 ΡίίτΛ¾¾?、 PiiraO? 和？^？^^等全启动子在结合缓冲液 （200 mM NaCl、 50mM Tris-HCl 8, 0、 10 mM MgCl2)中反应 40分钟。 用 12% TB- PAGE胶检测
DNA条带的迁移情况。 实验表明， 上述基因启动子均能结合 DdrO，发生条带迀移 (图 8 ) 。
(6) DdrO在体内结合 DNA损伤应急响应与修复基因启动子
运用染色质免疫共沉淀技术， 用 DdrO抗体纯化在体内与 DdrO蛋白结合的 DNA 序列， 再由 RT- PCR定量检测阴性对照基因 ^0¾？的丰度。 结果表明， 特异性的 DdiO抗体能富集所选基因启动子的数量与非特异性抗体的基本一致（图 9 ) 。
( 7 ) 在辐照处理恢复后期， 野生菌株 R1相对于 突变株 YR1胞内 DNA 损伤应急响应与修复基因转录水平趋于稳定
在 2kGy γ射线辐照处理后， 野生菌株 R1与 ^ 缺失菌株 YR1经恢复培养 90分钟离心收集，提取 RNA，反转录，用 qRT PCR检测体内 iirf?,、 dr2340、 dr2574、 dr()07(K dra0346, d:r()423、 drt^ ^^tl tir/ ^.*?等基因的转录水平， 其中
为明性基因， 处理前后表达量恒定。 结果表明， 在辐照处理恢复中后期， 野生 菌株 R1与 ¾ar/缺失菌株 YR1体内各个 DNA损伤应急响应与修复基因转录水平未发 生变化 （图 10， 图 1 1 )。
本发明实施例中所用的菌株为耐辐射奇球菌 ( Deinococcus radiodurans, ATCC No. 13939 ) , 但是根据本发明的教导和启示， 任何人工合成或者其他自然 含有的蛋白酶以及衍生物， 如具有与耐辐射奇球菌蛋白醇 Pprl同源的序列和类 似的结构和功能， 也在本发明的保护范围之内。

Claims

权 利 要 求 书
1、 一种蛋白酶， 其特征在于： 具有锌指-蛋白酶结构域、 螺旋-转角-螺旋结 构域和 GAF 结构域， 所述的蛋白酶全酶和独立的锌指-蛋白酶结构域具有同 等的蛋白酶活性。
2、 根据权利要求 1所述的蛋白酶， 其特征在于： 所述锌指-蛋白酶结构域核 心蛋白序列如 SEQ ID N0 : 1所示。
3、 根据权利要求 1 所述的蛋白酶， 其特征在于： 所述蛋白酶的底物之一为 耐福射奇球菌 Deinococcus radiodurans, ATCC No. 13939 的转录因子 DdrO (Gene ID : 1798752 ; NP— 296294. 1)，所述的转录因子可以在体内外结合耐辐 射奇球菌内含 RDRM位点的 DNA损伤应急响应与修复基因启动子。
4、 根据权利要求 1 所述的蛋白酶， 其特征在于： 所述蛋白酶的底物酶切特 异性序列是 ELXGXR ， 其中 X为任意一种必需氨基酸， 切割位置在第二和第 三个氨基酸残基之间。
5、 根据权利要求 1所述的蛋白酶， 其特征在于， 所述蛋白酶的酶切反应缓冲 液含 100-200mM NaCl、 10_50mM Tris-HCl 8. 0、 ImM DTT、 2. 0-5. 0 mM MnCl2
6、 根据权利要求 1 所述的蛋白酶， 其特征在于， 所述的蛋白酶的酶活性温 度范围为 4-65°C， 优选的所述蛋白酶的活性温度范围为 35-4CTC。
7、 根据权利要求 3所述的蛋白酶， 其特征在于， 所述的基因启动子包括 dr0070、 dr0099、 dra015 dr0219、 dr0326, dra0346, dr0423, dr0596、 dr0906、 drl039、 drll43、 drl289、 drl696, drl77 drl775、 drl913、 drl92 dr2256、 dr2275、 dr2336、 dr2574。
8、 根据权利要求 3所述的蛋白酶， 其特征在于， 与含 RDRM位点的基因启动 子的结合反应缓冲液含 100-200 mM NaCl、 20-50 mM Tris-HCl 8. 0、 5-10 mM MgCl2,反应温度为 30°C。
9、 根据权利要求 3所述的蛋白酶， 其特征在于， 所述的转录因子结合最小序 列为耐辐射奇球菌内的 RDRM位点。
10、 根据权利要求 1所述的蛋白酶， 其特征在于： 所述的蛋白酶来源于耐辐 射奇球菌。
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