CN114199848B - 一种基于蛋白连接酶的高通量检测蛋白表达的方法 - Google Patents

Abstract

本发明公开了一种基于蛋白连接酶的高通量检测蛋白表达的方法，涉及生物工程技术领域，将待测蛋白、蛋白连接酶和荧光多肽底物混合，利用蛋白连接酶连接携带识别序列的目的蛋白质和荧光多肽底物，获得待测蛋白表达检测结果，蛋白连接酶的氨基酸序列如SEQ ID NO.1所示。本发明利用识别PGA(15)基序的特异性蛋白连接酶(connectase)直接将荧光多肽底物连接到目标蛋白上，利用荧光标记的多肽底物在特定波长激发荧光下，可以即刻观察到目的蛋白表达情况，基于连接酶的方法可以更快速、灵敏、和特异地检测目的蛋白情况。

Description

一种基于蛋白连接酶的高通量检测蛋白表达的方法

技术领域

本发明涉及生物工程技术领域，具体涉及一种基于蛋白连接酶的高通量检测蛋白表达的方法。

背景技术

蛋白质是对生命至关重要的一类生物大分子物质，各种生命功能、生命现象、生命活动都与蛋白质有关。在生命有机体催化、运动、结构、识别和调节等多种方面，起着关键的作用。生物体内的天然蛋白获取方式困难，为了更好的研究蛋白对人类生命健康的影响，重组蛋白的产生已经成为生物医药产业中重要的一个方向。重组蛋白是使用重组DNA或重组RNA的技术而获得的蛋白质。体外生产主要包括四大系统：原核表达系统，昆虫表达系统、酵母表达系统及哺乳动物细胞表达系统。

重组蛋白表达后，一个最重要的过程就是检测蛋白是否表达。常用检测目的蛋白的方法主要有十二烷基硫酸钠聚丙烯酰胺凝胶电泳(SDS-PAGE)、蛋白质免疫印迹试验(Western blotting)等。其中SDS-PAGE检测蛋白表达情况时通量很低，过程繁琐，耗时较长，满足不了快速检测蛋白的需求。此外，不同表达系统在表达目的蛋白时，除表达目的蛋白外，还会内源性的蛋白表达，而且一些内源蛋白分子量与目的蛋白接近，在SDS-PAGE上会出现在同一位置，不能准确判断目的蛋白是否表达；当目标蛋白表达量很低时，SDS-PAGE也不能够检测出是否表达，这些情况下可能会出现判断不准确的现象。检测复杂溶液或活细胞中的蛋白质，通常通过荧光标记的抗体来完成，而抗体价格昂贵且需要特异性抗体，抗体的使用没有普遍性。

荧光检测灵敏性高、特异性强、耗时短、效率高。随着荧光标记技术的成熟和广泛应用，开发一种适用于荧光检测技术的目的蛋白快速检测方法显得尤为重要。基于上述内容，提出一种基于蛋白连接酶的高通量检测蛋白表达的方法。

发明内容

本发明的目的在于克服现有技术的不足，提供了一种识别PGA(15)基序的特异性蛋白连接酶及其应用。

本发明通过以下技术方案来实现上述目的：

本发明提供了一种基于蛋白连接酶的高通量检测蛋白表达的方法，主要是用一种与古菌甲基转移酶亚基A(MtrA)高度同源，能够特异性识别保守的PGA(15)基序的蛋白质连接酶，也可以通过人工合成，蛋白质连接酶识别荧光标记底物结合后直接将荧光标记在目标蛋白上，通过荧光信号强度检测蛋白表达情况，具体为将待测蛋白、蛋白连接酶和荧光多肽底物在反应缓冲液中混合，利用蛋白连接酶连接携带识别序列的目的蛋白质和荧光多肽底物，获得待测蛋白表达检测结果。

进一步改进在于，所述荧光多肽底物的氨基酸序列为依次连接的荧光素FITC、氨基己酸Ahx和PGA基序组成的氨基酸序列。

进一步改进在于，所述荧光多肽底物的氨基酸序列为：FITC-Ahx-RELASKDPGAFDADPLVVEISEEGE。

进一步改进在于，所述反应缓冲液为50mM HEPESpH7.0、150mM NaCl、50mM KCl、5mM TCEP,反应温度为37℃,反应时间为15min。

进一步改进在于，所述蛋白连接酶的氨基酸序列如SEQ ID NO.1所示。

本发明还提供了一种荧光多肽底物，所述荧光多肽底物的氨基酸序列为FITC-Ahx-RELASKDPGAFDADPLVVEISEEGE。

本发明还提供了一种蛋白连接酶，所述蛋白连接酶的氨基酸序列如SEQ ID NO.1所示。

本发明还提供了一种核苷酸序列，所述核苷酸序列为用于编码上述蛋白连接酶的氨基酸序列的核苷酸序列。

进一步改进在于，所述核苷酸序列如SEQ ID NO.2所示。

本发明还提供了一种蛋白表达载体，所述表达载体通过在pET-28a载体上插入上述的核苷酸序列获得。

本发明还提供了一种蛋白表达系统，为转入上述蛋白表达载体的大肠杆菌BL21菌株。

本发明具有如下有益效果：

1、本发明所述目标蛋白质检测方法即使在蛋白粗提溶液中，也能特异、有效连接携带识别序列的蛋白质，并将目的蛋白与荧光多肽底物相连接，消除了杂蛋白的干扰，并免去了使用亲和柱富集目的蛋白的过程，相比常用检测方法，既提高了效率又增加了通量。

2、本发明利用连接酶将荧光多肽连接到目的蛋白上，可通过荧光迁移变化显示有无表达目标蛋白，与蛋白质免疫印迹试验(Western blotting)相比，无需转膜、包被、孵探针等步骤，缩短了检测时间，且蛋白质免疫印迹试验探针价格昂贵，本发明中不需要使用探针，也大大节约了检测成本。

3、本发明所述目标蛋白质检测方法适用性广，可覆盖广泛分子量大小的蛋白，分子量范围可以从几kDa到100kDa以上大小的蛋白。

4、本发明所述方法，低成本快速的条件下，目标蛋白检测灵敏度达到纳克级别。

附图说明

图1为Connectase蛋白表达及纯化结果；

图2为Connectase蛋白质量检测结果；

图3为不同目标蛋白Tanon-5200荧光成像图；

图4为不同目标蛋白SDS-PAGE白光成像图；

图5为PGA(15)-6His-Ub为目的蛋白的Tanon-5200荧光成像图。

具体实施方式

下面结合附图对本申请作进一步详细描述，有必要在此指出的是，以下具体实施方式只用于对本申请进行进一步的说明，不能理解为对本申请保护范围的限制，该领域的技术人员可以根据上述申请内容对本申请作出一些非本质的改进和调整。

1、材料

本发明所用方法如无特别说明均为本领域的技术人员所知晓的常规方法，未注明具体条件者，按照常规条件或者制造商建议的条件进行，所用试剂或仪器未注明生产厂商者，均为可以通过市售购买获得的常规产品。

2、方法

2.1蛋白连接酶制备

2.1.1诱导表达融合蛋白

将编码蛋白连接酶的氨基酸序列的核苷酸序列克隆到pET-28a载体中，核苷酸序列如SEQ ID NO.2所示。从-80℃冰箱取出感受态细胞于冰上化冻，超净台内将表达载体使用常规分子生物学手段转化BL21大肠杆菌感受态细胞，42℃热击90s后将感受态细胞静置5分钟后，涂布到含有50μg/ml Amp+抗生素LB固体培养板上，在温度为37℃的培养箱中培养过夜，获得明显表达的菌株。

将明显表达的菌株接中至50mL LB液体培养基中37℃培养过夜，将过夜培养的细菌按1:100的比例接至5L LB液体培养基中，37℃培养至菌液OD600为0.6-0.8时加入0.5mMIPTG 15℃培养过夜，5000rpm离心收集菌体。

2.1.2蛋白纯化

将收集的菌块进行称重，按照1:10比例加入相应体积的裂解缓冲液(50mM Tris-HCl(pH 8.0),500mM NaCl,5％glycerol)，使用高压均质机破碎菌体，16000rpm高速离心收集上清。使用亲和层析His FF富集纯化蛋白，纯化前先用裂解buffer平衡His FF柱，将所有细胞上清挂柱后，用不同梯度的咪唑溶液洗脱，收集不同梯度咪唑洗脱下的蛋白进行SDS-PAGE检测，收集纯度较好的蛋白用Nanodrop测定蛋白浓度，计算蛋白产量。蛋白纯化结果如图1，根据纯化的结果显示连接酶(connectase)表达量高且纯度较好。

2.1.3蛋白质量检测

将咪唑洗脱下的蛋白取少量分别进行质谱和分子筛检测。质谱检测连接酶(connectase)的分子量为22920.1Da与其理论分子量23050.18Da非常接近，表明His FF纯化后的蛋白为目标蛋白，此外，分子筛的结果显示，连接酶在溶液中以单体的形式存在，大大地提高了其实际应用中的有效酶量。实验结果见图2。

2.2验证

为了验证连接酶检测蛋白表达的方法，用下列实施例进行验证。

本实施例中使用12种在大肠杆菌胞内表达具有不同分子量重组目标蛋白工程菌株进行验证，分别编号#1,#2,#3,#4,#5,#6,#7,#8,#9,#10,#11,#12，其表达目的蛋白的分子量的范围在15kDa-120kDa。编号#1-#12的氨基酸序列如SEQ ID NO.(3-14)所示。具体的实验方案如下。

2.2.1目标蛋白获取

将小量(10ml)表达的12种菌的菌体分别收集在1.5mL离心管富集菌体，加入1mL反应缓冲液：50mM HEPES pH7.0,150mM NaCl,50mM KCl,5mM TCEP，重悬细胞，使用超声波细胞破碎仪破碎细胞释放胞内蛋白。将1.5mL离心管置于低温离心机高速离心收集上清去除沉淀。

2.2.2制备样品

取50μL浓度为750nM蛋白连接酶至200μL离心管中，再加入50μL浓度为60μMFITC-PGA多肽底物(3176.47Da)，最后分别取50μL不同菌体裂解液上清加入200μL离心管中混匀启动反应，并标记#1,#2,#3,#4,#5,#6,#7,#8,#9,#10,#11,#12。将离心管置于37℃恒温培养箱中，反应15min，FITC-PGA多肽底物为一种荧光标记多肽底物，该底物可被蛋白连接酶特异性识别，荧光标记多肽底物(FITC-PGA(15)氨基酸序列为：FITC-Ahx-RELASKDPGAFDADPLVVEISEEGE。

2.2.3检测

取适量不同样品反应液加入等体积SDS-PAGE上样缓冲液，上样量20μL，电压200V，电泳30min。使用Tanon-5200选择荧光成像模式下绿色激发光源控制，获得成像，如图3所示，其中：

泳道1为阳性对照蛋白Ub-PGA(MW:11kDa)；

泳道2为样品#1(MW:16kDa)；

泳道3为样品#2(MW:28kDa)；

泳道4为样品#3(MW:40kDa)；

泳道5为样品#4(MW:42kDa)；

泳道6为样品#5(MW:53kDa)；

泳道7为样品#6(MW:62kDa)；

泳道8为样品#7(MW:70kDa)；

泳道9为样品#8(MW:85kDa)；

泳道10为样品#9(MW:87kDa)；

泳道11为样品#10(MW:95kDa)；

泳道12为样品#11(MW:108kDa)；

泳道13为样品#12(MW:121kDa)。

由图可见，分子量10-120kDa范围内的蛋白均可被检测，说明该方法对蛋白分子量无选择性，适用于大规模广泛筛选。同时将该SDS-PAGE用白光进行成像，结果如图4，从白光成像的结果来看，12种不同的目的蛋白均表达，且位置大小与荧光成像位置相同。

对比两个结果可以看到，普通白光成像中目标蛋白与其他内源性蛋白均在上清液中被检测到，而在荧光成像中，目标蛋白特异性很好，仅目标蛋白被标记上荧光，说明该方法特异性好、大大地降低了实验误差。此外，从#10、#11和#12菌株表达蛋白的荧光成像结果可知，目标蛋白还存在降解条带，这在普通的SDS-PAGE白光成像中不能被检测，该方法还在蛋白稳定性发明提供了重要信息，为后续蛋白的制备提供了帮助。

2.2.4进一步验证

以PGA(15)-6His-Ub为目的蛋白，检测的蛋白含量从10μg起始，按照2倍梯度稀释比例，共稀释10个梯度，加入到不同的反应管中，Control为在反应体系中不加PGA(15)-6His-Ub，分别在每个反应管中下加入0.635μg FITC-PGA多肽底物和0.057μg蛋白连接酶，置于37℃恒温培养箱中，反应15min，SDS-PAGE检测，结果如图5所示，Tanon-5200荧光成像模式下，曝光6s，可检测最低20ng的目的蛋白。本实施例鉴定了蛋白连接酶检测蛋白的最低量为20ng，说明本发明可以检测很少量蛋白的表达，解决了目标蛋白因表达量低而检测不到的现象，提高了蛋白检测的效率和准确性。

以上所述实施例仅表达了本发明的几种实施方式，其描述较为具体和详细，但并不能因此而理解为对本发明专利范围的限制。应当指出的是，对于本领域的普通技术人员来说，在不脱离本发明构思的前提下，还可以做出若干变形和改进，这些都属于本发明的保护范围。

序列表

<110> 无锡佰翱得生物科学有限公司

<120> 一种基于蛋白连接酶的高通量检测蛋白表达的方法

<160> 14

<170> SIPOSequenceListing 1.0

<210> 1

<211> 208

<212> PRT

<213> 古菌甲基转移酶亚基A(MtrA)

<400> 1

Met Thr Leu Val Ile Ala Phe Ile Gly Lys Asn Gly Ala Val Met Ala

1 5 10 15

Gly Asp Met Arg Glu Ile Thr Phe Glu Gly Glu Lys Pro Asp Arg Glu

20 25 30

Lys Leu Glu Lys Glu Leu Tyr Ser Gly Ser Ile Val Thr Asp Glu Glu

35 40 45

Met Gln Lys Lys Ala Glu Glu Phe Gly Val Lys Ile Thr Val Ala Asp

50 55 60

Cys Lys Glu Lys Val Ser Glu Arg Asn Gly Val Leu Val Gly Glu Val

65 70 75 80

Ser Ser Ala Glu Gly Gly Val Val Lys Lys Arg Arg Leu Tyr Ala Ser

85 90 95

Ala Gly Asn Phe Ala Ile Ala Glu Leu Ile Asn Thr Glu Met Thr Leu

100 105 110

Thr Ser Gln Gly Lys Gly Ser Asn Phe Ile Ala Phe Gly Asn Glu Phe

115 120 125

Thr Lys Gln Val Ala Asn Lys Cys Phe Lys Asp Asn Trp Thr Lys Lys

130 135 140

Ser Asn Leu Gln Asp Ala Val Lys Ile Leu Ile Leu Cys Met Glu Thr

145 150 155 160

Val Ala Arg Lys Thr Ala Ser Val Ser Lys Gln Phe Met Ile Val Gln

165 170 175

Thr Ala Ser Asn Ala Asp Val Leu Lys Val Val Glu Lys Asp Arg Asn

180 185 190

Cys Gly Ser Glu Asn Leu Tyr Phe Gln Gly His His His His His His

195 200 205

<210> 2

<211> 630

<212> DNA

<213> 古菌甲基转移酶亚基A(MtrA)

<400> 2

atgacactag taatagcttt tattggaaaa aacggcgctg tgatggcagg cgacatgcgt 60

gaaattacgt ttgaaggtga gaagccggat cgcgagaagc tggaaaagga gttgtacagc 120

ggtagtatcg ttaccgatga agagatgcag aaaaaggctg aggagttcgg cgtgaaaatc 180

acggtggcgg actgtaaaga gaaagttagc gaacgtaatg gcgtcttggt tggtgaagtt 240

tcgtccgcag agggcggcgt cgtgaagaaa agacgtctgt acgcgtccgc gggtaatttt 300

gcaattgcgg agctgatcaa caccgagatg accctgacca gccagggtaa aggttctaac 360

ttcatcgcct ttggcaatga attcaccaag caagttgcta ataagtgctt caaagacaac 420

tggaccaaga aaagcaacct gcaagatgcg gtgaagatct taattctttg catggaaacg 480

gttgcgcgta aaactgcgag cgttagcaaa caatttatga ttgtacagac cgcctctaac 540

gccgatgtcc tgaaagtggt ggaaaaggac cgcaactgcg gttccgaaaa cctgtatttc 600

cagggtcatc accaccatca ccactaatga 630

<210> 3

<211> 144

<212> PRT

<213> 古菌甲基转移酶亚基A(MtrA)

<400> 3

Met Pro Gly Ala Phe Asp Ala Asp Pro Leu Val Val Glu Ile Ser Glu

1 5 10 15

Glu Gly Glu Met Gly Ser Ser His His His His His His Glu Asn Leu

20 25 30

Tyr Phe Gln Gly Gly Gly Ala Asp Leu Glu Asp Asn Trp Glu Thr Leu

35 40 45

Asn Asp Asn Leu Lys Val Ile Glu Lys Ala Asp Asn Ala Ala Gln Val

50 55 60

Lys Asp Ala Leu Thr Lys Met Arg Ala Ala Ala Leu Asp Ala Gln Lys

65 70 75 80

Ala Thr Pro Pro Lys Leu Glu Asp Lys Ser Pro Asp Ser Pro Glu Met

85 90 95

Trp Asp Phe Arg His Gly Phe Asp His Leu Val Tyr His Ile Asp Asp

100 105 110

Ala Leu Lys Leu Ala Asn Glu Gly Lys Val Lys Glu Ala Gln Ala Ala

115 120 125

Ala Glu Gln Leu Lys Cys His Arg Asn Ala Ala Ile Gln Lys Tyr Leu

130 135 140

<210> 4

<211> 246

<212> PRT

<213> 古菌甲基转移酶亚基A(MtrA)

<400> 4

Met Pro Gly Ala Phe Asp Ala Asp Pro Leu Val Val Glu Ile Ser Glu

1 5 10 15

Glu Gly Glu Met Arg Tyr Tyr Glu Lys Ile Asp Gly Ser Lys Tyr Arg

20 25 30

Asn Ile Trp Val Val Gly Asp Leu His Gly Cys Tyr Thr Asn Leu Met

35 40 45

Asn Lys Leu Asp Thr Ile Gly Phe Asp Asn Lys Lys Asp Leu Leu Ile

50 55 60

Ser Val Gly Asp Leu Val Asp Arg Gly Ala Glu Asn Val Glu Cys Leu

65 70 75 80

Glu Leu Ile Thr Phe Pro Trp Phe Arg Ala Val Arg Gly Asn His Glu

85 90 95

Gln Met Met Ile Asp Gly Leu Ser Glu Arg Gly Asn Val Asn His Trp

100 105 110

Leu Leu Asn Gly Gly Gly Trp Phe Phe Asn Leu Asp Tyr Asp Lys Glu

115 120 125

Ile Leu Ala Lys Ala Leu Ala His Lys Ala Asp Glu Leu Pro Leu Ile

130 135 140

Ile Glu Leu Val Ser Lys Asp Lys Lys Tyr Val Ile Cys His Ala Asp

145 150 155 160

Tyr Pro Phe Asp Glu Tyr Glu Phe Gly Lys Pro Val Asp His Gln Gln

165 170 175

Val Ile Trp Asn Arg Glu Arg Ile Ser Asn Ser Gln Asn Gly Ile Val

180 185 190

Lys Glu Ile Lys Gly Ala Asp Thr Phe Ile Phe Gly His Thr Pro Ala

195 200 205

Val Lys Pro Leu Lys Phe Ala Asn Gln Met Tyr Ile Asp Thr Gly Ala

210 215 220

Val Phe Cys Gly Asn Leu Thr Leu Ile Gln Val Gln Gly Glu Gly Ala

225 230 235 240

His His His His His His

245

<210> 5

<211> 360

<212> PRT

<213> 古菌甲基转移酶亚基A(MtrA)

<400> 5

Met Pro Gly Ala Phe Asp Ala Asp Pro Leu Val Val Glu Ile Ser Glu

1 5 10 15

Glu Gly Glu Met Gly Ser Ser His His His His His His Ser Asp Ser

20 25 30

Glu Val Asn Gln Glu Ala Lys Pro Glu Val Lys Pro Glu Val Lys Pro

35 40 45

Glu Thr His Ile Asn Leu Lys Val Ser Asp Gly Ser Ser Glu Ile Phe

50 55 60

Phe Lys Ile Lys Lys Thr Thr Pro Leu Arg Arg Leu Met Glu Ala Phe

65 70 75 80

Ala Lys Arg Gln Gly Lys Glu Met Asp Ser Leu Arg Phe Leu Tyr Asp

85 90 95

Gly Ile Arg Ile Gln Ala Asp Gln Thr Pro Glu Asp Leu Asp Met Glu

100 105 110

Asp Asn Asp Ile Ile Glu Ala His Arg Glu Gln Ile Gly Gly Ile Leu

115 120 125

Val Glu Gly Asp Gly Ile Pro Pro Pro Ile Lys Ser Phe Lys Glu Met

130 135 140

Lys Phe Pro Ala Ala Ile Leu Arg Gly Leu Lys Lys Lys Gly Ile His

145 150 155 160

His Pro Thr Pro Ile Gln Ile Gln Gly Ile Pro Thr Ile Leu Ser Gly

165 170 175

Arg Asp Met Ile Gly Ile Ala Phe Thr Gly Ser Gly Lys Thr Leu Val

180 185 190

Phe Thr Leu Pro Val Ile Met Phe Cys Leu Glu Gln Glu Lys Arg Leu

195 200 205

Pro Phe Ser Lys Arg Glu Gly Pro Tyr Gly Leu Ile Ile Cys Pro Ser

210 215 220

Arg Glu Leu Ala Arg Gln Thr His Gly Ile Leu Glu Tyr Tyr Cys Arg

225 230 235 240

Leu Leu Gln Glu Asp Ser Ser Pro Leu Leu Arg Cys Ala Leu Cys Ile

245 250 255

Gly Gly Met Ser Val Lys Glu Gln Met Glu Thr Ile Arg His Gly Val

260 265 270

His Met Met Val Ala Thr Pro Gly Arg Leu Met Asp Leu Leu Gln Lys

275 280 285

Lys Met Val Ser Leu Asp Ile Cys Arg Tyr Leu Ala Leu Asp Glu Ala

290 295 300

Asp Arg Met Ile Asp Met Gly Phe Glu Gly Asp Ile Arg Thr Ile Phe

305 310 315 320

Ser Tyr Phe Lys Gly Gln Arg Gln Thr Leu Leu Phe Ser Ala Thr Met

325 330 335

Pro Lys Lys Ile Gln Asn Phe Ala Lys Ser Ala Leu Val Lys Pro Val

340 345 350

Thr Ile Asn Val Gly Arg Ala Gly

355 360

<210> 6

<211> 385

<212> PRT

<213> 古菌甲基转移酶亚基A(MtrA)

<400> 6

Met Pro Gly Ala Phe Asp Ala Asp Pro Leu Val Val Glu Ile Ser Glu

1 5 10 15

Glu Gly Glu Met Gly Ser Ser His His His His His His Glu Asn Leu

20 25 30

Tyr Phe Gln Gly Gly Gly Met Gly Phe Gln Glu Val Tyr Gly Val Leu

35 40 45

Pro Gln Ala Ser Ala Gln Ala Pro Gly Arg Val Asn Leu Leu Gly Glu

50 55 60

His Thr Asp Tyr Gln Glu Gly Tyr Val Leu Pro Thr Pro Val Pro Tyr

65 70 75 80

Phe Thr Arg Val Glu Ala Ala Pro Leu Glu Gly Ala Val Glu Ala Phe

85 90 95

Ser Glu Asn Leu Gly Glu Leu Arg Ala Arg Pro Leu Ser Ser Pro Pro

100 105 110

Gln Gly Asp Phe Leu Asp Tyr Leu Leu Gly Val Val Arg Ala Leu Arg

115 120 125

Glu Ala Gly His Gly Val Glu Gly Ala Arg Phe Tyr Val Arg Ser Asp

130 135 140

Leu Pro Met Gly Ala Gly Leu Ser Ser Ser Ala Ala Leu Glu Val Ala

145 150 155 160

Ala Leu Arg Ala Leu Arg Thr Leu Tyr Arg Leu Pro Leu Ser Asp Leu

165 170 175

Glu Val Ala Arg Leu Ala Gln Lys Ala Glu Val Glu Tyr Val Gly Val

180 185 190

Arg Cys Gly Ile Met Asp Gln Met Ala Ala Ser Leu Gly Gln Pro Gly

195 200 205

Gln Ala Leu Phe Leu Asp Thr Arg Thr Leu Ala Tyr Glu Asn Leu Pro

210 215 220

Leu Pro Pro Gly Val Arg Val Ala Val Leu Asp Leu Gly Leu Gly Arg

225 230 235 240

Arg Leu Ala Glu Ala Gly Tyr Asn Gln Arg Arg Gln Glu Ala Glu Glu

245 250 255

Ala Ala Lys Arg Leu Gly Val Arg Ser Leu Arg Asp Val Ala Asp Leu

260 265 270

Cys Leu Val Glu Ser Leu Pro Ser Pro Leu Asp Arg Arg Ala Arg His

275 280 285

Val Val Ser Glu Asn Leu Arg Val Leu Arg Gly Val Glu Ala Leu Arg

290 295 300

Arg Gly Asp Ala Arg Ala Phe Gly Glu Leu Met Thr Ala Ser His Arg

305 310 315 320

Ser Leu Ala Gln Asp Tyr Glu Val Ser Leu Pro Glu Leu Asp Leu Leu

325 330 335

Val Glu Glu Ala Leu Lys Ala Gly Ala Tyr Gly Ala Lys Leu Thr Gly

340 345 350

Ala Gly Phe Gly Gly Ala Val Val Ala Leu Val Ala Glu Ser Arg Phe

355 360 365

Pro Ala Phe Arg Glu Ala Leu Ala Arg Arg Phe Pro Asp Leu Lys Val

370 375 380

Leu

385

<210> 7

<211> 506

<212> PRT

<213> 古菌甲基转移酶亚基A(MtrA)

<400> 7

Met Pro Gly Ala Phe Asp Ala Asp Pro Leu Val Val Glu Ile Ser Glu

1 5 10 15

Glu Gly Glu Met Gly Gly Ser His His His His His His Glu Asn Leu

20 25 30

Tyr Phe Gln Gly Asp Ala Asp Leu Trp Ser Ser His Asp Ala Met Leu

35 40 45

Ala Gln Pro Leu Lys Asp Ser Asp Val Glu Val Tyr Asn Ile Ile Lys

50 55 60

Lys Glu Ser Asn Arg Gln Arg Val Gly Leu Glu Leu Ile Ala Ser Glu

65 70 75 80

Asn Phe Ala Ser Arg Ala Val Leu Glu Ala Leu Gly Ser Cys Leu Asn

85 90 95

Asn Lys Tyr Ser Glu Gly Tyr Pro Gly Gln Arg Tyr Tyr Gly Gly Thr

100 105 110

Glu Phe Ile Asp Glu Leu Glu Thr Leu Cys Gln Lys Arg Ala Leu Gln

115 120 125

Ala Tyr Lys Leu Asp Pro Gln Cys Trp Gly Val Asn Val Gln Pro Tyr

130 135 140

Ser Gly Ser Pro Ala Asn Phe Ala Val Tyr Thr Ala Leu Val Glu Pro

145 150 155 160

His Gly Arg Ile Met Gly Leu Asp Leu Pro Asp Gly Gly His Leu Thr

165 170 175

His Gly Phe Met Thr Asp Lys Lys Lys Ile Ser Ala Thr Ser Ile Phe

180 185 190

Phe Glu Ser Met Pro Tyr Lys Val Asn Pro Asp Thr Gly Tyr Ile Asn

195 200 205

Tyr Asp Gln Leu Glu Glu Asn Ala Arg Leu Phe His Pro Lys Leu Ile

210 215 220

Ile Ala Gly Thr Ser Cys Tyr Ser Arg Asn Leu Glu Tyr Ala Arg Leu

225 230 235 240

Arg Lys Ile Ala Asp Glu Asn Gly Ala Tyr Leu Met Ala Asp Met Ala

245 250 255

His Ile Ser Gly Leu Val Ala Ala Gly Val Val Pro Ser Pro Phe Glu

260 265 270

His Cys His Val Val Thr Thr Thr Thr His Lys Thr Leu Arg Gly Cys

275 280 285

Arg Ala Gly Met Ile Phe Tyr Arg Lys Gly Val Lys Ser Val Asp Pro

290 295 300

Ala Thr Gly Lys Glu Ile Leu Tyr Asn Leu Glu Ser Leu Ile Asn Ser

305 310 315 320

Ala Val Phe Pro Gly Leu Gln Gly Gly Pro His Asn His Ala Ile Ala

325 330 335

Gly Val Ala Val Ala Leu Lys Gln Ala Met Thr Leu Glu Phe Lys Val

340 345 350

Tyr Gln His Gln Val Val Ala Asn Cys Arg Ala Leu Ser Glu Ala Leu

355 360 365

Thr Glu Leu Gly Tyr Lys Ile Val Thr Gly Gly Ser Asp Asn His Leu

370 375 380

Ile Leu Val Asp Leu Arg Ser Lys Gly Thr Asp Gly Gly Arg Ala Glu

385 390 395 400

Lys Val Leu Glu Ala Cys Ser Ile Ala Cys Asn Lys Asn Thr Cys Pro

405 410 415

Gly Asp Arg Ser Ala Leu Arg Pro Ser Gly Leu Arg Leu Gly Thr Pro

420 425 430

Ala Leu Thr Ser Arg Gly Leu Leu Glu Lys Asp Phe Gln Lys Val Ala

435 440 445

His Phe Ile His Arg Gly Ile Glu Leu Thr Leu Gln Ile Gln Ser Asp

450 455 460

Thr Gly Val Ala Ala Thr Leu Lys Glu Phe Lys Glu Arg Leu Ala Gly

465 470 475 480

Asp Lys Tyr Gln Ala Ala Val Gln Ala Leu Arg Glu Glu Val Glu Ser

485 490 495

Phe Ala Ser Leu Phe Pro Leu Pro Gly Leu

500 505

<210> 8

<211> 561

<212> PRT

<213> 古菌甲基转移酶亚基A(MtrA)

<400> 8

Met Pro Gly Ala Phe Asp Ala Asp Pro Leu Val Val Glu Ile Ser Glu

1 5 10 15

Glu Gly Glu Met Gly Gly Ser His His His His His His Glu Asn Leu

20 25 30

Tyr Phe Gln Gly Met Lys Ser Arg Arg Trp Phe His Pro Asn Ile Thr

35 40 45

Gly Val Glu Ala Glu Asn Leu Leu Leu Thr Arg Gly Val Asp Gly Ser

50 55 60

Phe Leu Ala Arg Pro Ser Lys Ser Asn Pro Gly Asp Leu Thr Leu Ser

65 70 75 80

Val Arg Arg Asn Gly Ala Val Thr His Ile Lys Ile Gln Asn Thr Gly

85 90 95

Asp Tyr Tyr Asp Leu Tyr Gly Gly Glu Lys Phe Ala Thr Leu Ala Glu

100 105 110

Leu Val Gln Tyr Tyr Met Glu His His Gly Gln Leu Lys Glu Lys Asn

115 120 125

Gly Asp Val Ile Glu Leu Lys Tyr Pro Leu Asn Cys Ala Asp Pro Thr

130 135 140

Ser Glu Arg Trp Phe His Gly His Leu Ser Gly Lys Glu Ala Glu Lys

145 150 155 160

Leu Leu Thr Glu Lys Gly Lys His Gly Ser Phe Leu Val Arg Glu Ser

165 170 175

Gln Ser His Pro Gly Asp Phe Val Leu Ser Val Arg Thr Gly Asp Asp

180 185 190

Lys Gly Glu Ser Asn Asp Gly Lys Ser Lys Val Thr His Val Met Ile

195 200 205

Arg Cys Gln Glu Leu Lys Tyr Asp Val Gly Gly Gly Glu Arg Phe Asp

210 215 220

Ser Leu Thr Asp Leu Val Glu His Tyr Lys Lys Asn Pro Met Val Glu

225 230 235 240

Thr Leu Gly Thr Val Leu Gln Leu Lys Gln Pro Leu Asn Thr Thr Arg

245 250 255

Ile Asn Ala Ala Glu Ile Glu Ser Arg Val Arg Glu Leu Ser Lys Leu

260 265 270

Ala Glu Thr Thr Asp Lys Val Lys Gln Gly Phe Trp Glu Glu Phe Glu

275 280 285

Thr Leu Gln Gln Gln Glu Cys Lys Leu Leu Tyr Ser Arg Lys Glu Gly

290 295 300

Gln Arg Gln Glu Asn Lys Asn Lys Asn Arg Tyr Lys Asn Ile Leu Pro

305 310 315 320

Phe Asp His Thr Arg Val Val Leu His Asp Gly Asp Pro Asn Glu Pro

325 330 335

Val Ser Asp Tyr Ile Asn Ala Asn Ile Ile Met Pro Glu Phe Glu Thr

340 345 350

Lys Cys Asn Asn Ser Lys Pro Lys Lys Ser Tyr Ile Ala Thr Gln Gly

355 360 365

Cys Leu Gln Asn Thr Val Asn Asp Phe Trp Arg Met Val Phe Gln Glu

370 375 380

Asn Ser Arg Val Ile Val Met Thr Thr Lys Glu Val Glu Arg Gly Lys

385 390 395 400

Ser Lys Cys Val Lys Tyr Trp Pro Asp Glu Tyr Ala Leu Lys Glu Tyr

405 410 415

Gly Val Met Arg Val Arg Asn Val Lys Glu Ser Ala Ala His Asp Tyr

420 425 430

Thr Leu Arg Glu Leu Lys Leu Ser Lys Val Gly Gln Gly Asn Thr Glu

435 440 445

Arg Thr Val Trp Gln Tyr His Phe Arg Thr Trp Pro Asp His Gly Val

450 455 460

Pro Ser Asp Pro Gly Gly Val Leu Asp Phe Leu Glu Glu Val His His

465 470 475 480

Lys Gln Glu Ser Ile Met Asp Ala Gly Pro Val Val Val His Cys Ser

485 490 495

Ala Gly Ile Gly Arg Thr Gly Thr Phe Ile Val Ile Asp Ile Leu Ile

500 505 510

Asp Ile Ile Arg Glu Lys Gly Val Asp Cys Asp Ile Asp Val Pro Lys

515 520 525

Thr Ile Gln Met Val Arg Ser Gln Arg Ser Gly Met Val Gln Thr Glu

530 535 540

Ala Gln Tyr Arg Ser Ile Tyr Met Ala Val Gln His Tyr Ile Glu Thr

545 550 555 560

Leu

<210> 9

<211> 629

<212> PRT

<213> 古菌甲基转移酶亚基A(MtrA)

<400> 9

Met Pro Gly Ala Phe Asp Ala Asp Pro Leu Val Val Glu Ile Ser Glu

1 5 10 15

Glu Gly Glu Met Asp Tyr Lys Asp Asp Asp Asp Lys Glu Asn Leu Tyr

20 25 30

Phe Gln Gly Met Gln Ile Phe Val Lys Thr Leu Thr Gly Lys Thr Ile

35 40 45

Thr Leu Glu Val Glu Pro Ser Asp Thr Ile Glu Asn Val Lys Ala Lys

50 55 60

Ile Gln Asp Lys Glu Gly Ile Pro Pro Asp Gln Gln Arg Leu Ile Phe

65 70 75 80

Ala Gly Lys Gln Leu Glu Asp Gly Arg Thr Leu Ser Asp Tyr Asn Ile

85 90 95

Gln Lys Glu Ser Thr Leu His Leu Val Leu Arg Leu Arg Gly Pro Gly

100 105 110

Cys Phe Ala Lys Gly Thr Asn Val Leu Met Ala Asp Gly Ser Ile Glu

115 120 125

Cys Ile Glu Asn Ile Glu Val Gly Asn Lys Val Met Gly Lys Asp Gly

130 135 140

Arg Pro Arg Glu Val Ile Lys Leu Pro Arg Gly Arg Glu Thr Met Tyr

145 150 155 160

Ser Val Val Gln Lys Ser Gln His Arg Ala His Lys Ser Asp Ser Ser

165 170 175

Arg Glu Val Pro Glu Leu Leu Lys Phe Thr Cys Asn Ala Thr His Glu

180 185 190

Leu Val Val Arg Thr Pro Arg Ser Val Arg Arg Leu Ser Arg Thr Ile

195 200 205

Lys Gly Val Glu Tyr Phe Glu Val Ile Thr Phe Glu Met Gly Gln Lys

210 215 220

Lys Ala Pro Asp Gly Arg Ile Val Glu Leu Val Lys Glu Val Ser Lys

225 230 235 240

Ser Tyr Pro Ile Ser Glu Gly Pro Glu Arg Ala Asn Glu Leu Val Glu

245 250 255

Ser Tyr Arg Lys Ala Ser Asn Lys Ala Tyr Phe Glu Trp Thr Ile Glu

260 265 270

Ala Arg Asp Leu Ser Leu Leu Gly Ser His Val Arg Lys Ala Thr Tyr

275 280 285

Gln Thr Tyr Ala Pro Ile Leu Tyr Glu Asn Asp His Phe Phe Asp Tyr

290 295 300

Met Gln Lys Ser Lys Phe His Leu Thr Ile Glu Gly Pro Lys Val Leu

305 310 315 320

Ala Tyr Leu Leu Gly Leu Trp Ile Gly Asp Gly Leu Ser Asp Arg Ala

325 330 335

Thr Phe Ser Val Asp Ser Arg Asp Thr Ser Leu Met Glu Arg Val Thr

340 345 350

Glu Tyr Ala Glu Lys Leu Asn Leu Cys Ala Glu Tyr Lys Asp Arg Lys

355 360 365

Glu Pro Gln Val Ala Lys Thr Val Asn Leu Tyr Ser Lys Val Val Arg

370 375 380

Gly Asn Gly Ile Arg Asn Asn Leu Asn Thr Glu Asn Pro Leu Trp Asp

385 390 395 400

Ala Ile Val Gly Leu Gly Phe Leu Lys Asp Gly Val Lys Asn Ile Pro

405 410 415

Ser Phe Leu Ser Thr Asp Asn Ile Gly Thr Arg Glu Thr Phe Leu Ala

420 425 430

Gly Leu Ile Asp Ser Asp Gly Tyr Val Thr Asp Glu His Gly Ile Lys

435 440 445

Ala Thr Ile Lys Thr Ile His Thr Ser Val Arg Asp Gly Leu Val Ser

450 455 460

Leu Ala Arg Ser Leu Gly Leu Val Val Ser Val Asn Ala Glu Pro Ala

465 470 475 480

Lys Val Asp Met Asn Val Thr Lys His Lys Ile Ser Tyr Ala Ile Tyr

485 490 495

Met Ser Gly Gly Asp Val Leu Leu Asn Val Leu Ser Lys Cys Ala Gly

500 505 510

Ser Lys Lys Phe Arg Pro Ala Pro Ala Ala Ala Phe Ala Arg Glu Cys

515 520 525

Arg Gly Phe Tyr Phe Glu Leu Gln Glu Leu Lys Glu Asp Asp Tyr Tyr

530 535 540

Gly Ile Thr Leu Ser Asp Asp Ser Asp His Gln Phe Leu Leu Gly Ser

545 550 555 560

Gln Val Val Val His Ala Cys Gly Gly Leu Thr Gly Leu Asn Ser Gly

565 570 575

Leu Thr Thr Asn Pro Gly Val Ser Ala Trp Gln Val Asn Thr Ala Tyr

580 585 590

Thr Ala Gly Gln Leu Val Thr Tyr Asn Gly Lys Thr Tyr Lys Cys Leu

595 600 605

Gln Pro His Thr Ser Leu Ala Gly Trp Glu Pro Ser Asn Val Pro Ala

610 615 620

Leu Trp Gln Leu Gln

625

<210> 10

<211> 752

<212> PRT

<213> 古菌甲基转移酶亚基A(MtrA)

<400> 10

Met Pro Gly Ala Phe Asp Ala Asp Pro Leu Val Val Glu Ile Ser Glu

1 5 10 15

Glu Gly Glu Met His His His His His His Glu Asn Leu Tyr Phe Gln

20 25 30

Gly Met Phe Gly Lys Lys Lys Lys Lys Ile Glu Ile Ser Gly Pro Ser

35 40 45

Asn Phe Glu His Arg Val His Thr Gly Phe Asp Pro Gln Glu Gln Lys

50 55 60

Phe Thr Gly Leu Pro Gln Gln Trp His Ser Leu Leu Ala Asp Thr Ala

65 70 75 80

Asn Arg Pro Lys Pro Met Val Asp Pro Ser Cys Ile Thr Pro Ile Gln

85 90 95

Leu Ala Pro Met Lys Thr Ile Val Arg Gly Asn Lys Pro Cys Lys Glu

100 105 110

Thr Ser Ile Asn Gly Leu Leu Glu Asp Phe Asp Asn Ile Ser Val Thr

115 120 125

Arg Ser Asn Ser Leu Arg Lys Glu Ser Pro Pro Thr Pro Asp Gln Gly

130 135 140

Ala Ser Ser His Gly Pro Gly His Ala Glu Glu Asn Gly Phe Ile Thr

145 150 155 160

Phe Ser Gln Tyr Ser Ser Glu Ser Asp Thr Thr Ala Asp Tyr Thr Thr

165 170 175

Glu Lys Tyr Arg Glu Lys Ser Leu Tyr Gly Asp Asp Leu Asp Pro Tyr

180 185 190

Tyr Arg Gly Ser His Ala Ala Lys Gln Asn Gly His Val Met Lys Met

195 200 205

Lys His Gly Glu Ala Tyr Tyr Ser Glu Val Lys Pro Leu Lys Ser Asp

210 215 220

Phe Ala Arg Phe Ser Ala Asp Tyr His Ser His Leu Asp Ser Leu Ser

225 230 235 240

Lys Pro Ser Glu Tyr Ser Asp Leu Lys Trp Glu Tyr Gln Arg Ala Ser

245 250 255

Ser Ser Ser Pro Leu Asp Tyr Ser Phe Gln Phe Thr Pro Ser Arg Thr

260 265 270

Ala Gly Thr Ser Gly Cys Ser Lys Glu Ser Leu Ala Tyr Ser Glu Ser

275 280 285

Glu Trp Gly Pro Ser Leu Asp Asp Tyr Asp Arg Arg Pro Lys Ser Ser

290 295 300

Tyr Leu Asn Gln Thr Ser Pro Gln Pro Thr Met Arg Gln Arg Ser Arg

305 310 315 320

Ser Gly Ser Gly Leu Gln Glu Pro Met Met Pro Phe Gly Ala Ser Ala

325 330 335

Phe Lys Thr His Pro Gln Gly His Ser Tyr Asn Ser Tyr Thr Tyr Pro

340 345 350

Arg Leu Ser Glu Pro Thr Met Cys Ile Pro Lys Val Asp Tyr Asp Arg

355 360 365

Ala Gln Met Val Leu Ser Pro Pro Leu Ser Gly Ser Asp Thr Tyr Pro

370 375 380

Arg Gly Pro Ala Lys Leu Pro Gln Ser Gln Ser Lys Ser Gly Tyr Ser

385 390 395 400

Ser Ser Ser His Gln Tyr Pro Ser Gly Tyr His Lys Ala Thr Leu Tyr

405 410 415

His His Pro Ser Leu Gln Ser Ser Ser Gln Tyr Ile Ser Thr Ala Ser

420 425 430

Tyr Leu Ser Ser Leu Ser Leu Ser Ser Ser Thr Tyr Pro Pro Pro Ser

435 440 445

Trp Gly Ser Ser Ser Asp Gln Gln Pro Ser Arg Val Ser His Glu Gln

450 455 460

Phe Arg Ala Ala Leu Gln Leu Val Val Ser Pro Gly Asp Pro Arg Glu

465 470 475 480

Tyr Leu Ala Asn Phe Ile Lys Ile Gly Glu Gly Ser Thr Gly Ile Val

485 490 495

Cys Ile Ala Thr Glu Lys His Thr Gly Lys Gln Val Ala Val Lys Lys

500 505 510

Met Asp Leu Arg Lys Gln Gln Arg Arg Glu Leu Leu Phe Asn Glu Val

515 520 525

Val Ile Met Arg Asp Tyr His His Asp Asn Val Val Asp Met Tyr Ser

530 535 540

Ser Tyr Leu Val Gly Asp Glu Leu Trp Val Val Met Glu Phe Leu Glu

545 550 555 560

Gly Gly Ala Leu Thr Asp Ile Val Thr His Thr Arg Met Asn Glu Glu

565 570 575

Gln Ile Ala Thr Val Cys Leu Ser Val Leu Arg Ala Leu Ser Tyr Leu

580 585 590

His Asn Gln Gly Val Ile His Arg Asp Ile Lys Ser Asp Ser Ile Leu

595 600 605

Leu Thr Ser Asp Gly Arg Ile Lys Leu Ser Asp Phe Gly Phe Cys Ala

610 615 620

Gln Val Ser Lys Glu Val Pro Lys Arg Lys Ser Leu Val Gly Thr Pro

625 630 635 640

Tyr Trp Met Ala Pro Glu Val Ile Ser Arg Leu Pro Tyr Gly Thr Glu

645 650 655

Val Asp Ile Trp Ser Leu Gly Ile Met Val Ile Glu Met Ile Asp Gly

660 665 670

Glu Pro Pro Tyr Phe Asn Glu Pro Pro Leu Gln Ala Met Arg Arg Ile

675 680 685

Arg Asp Ser Leu Pro Pro Arg Val Lys Asp Leu His Lys Val Ser Ser

690 695 700

Val Leu Arg Gly Phe Leu Asp Leu Met Leu Val Arg Glu Pro Ser Gln

705 710 715 720

Arg Ala Thr Ala Gln Glu Leu Leu Gly His Pro Phe Leu Lys Leu Ala

725 730 735

Gly Pro Pro Ser Cys Ile Val Pro Leu Met Arg Gln Tyr Arg His His

740 745 750

<210> 11

<211> 774

<212> PRT

<213> 古菌甲基转移酶亚基A(MtrA)

<400> 11

Met Pro Gly Ala Phe Asp Ala Asp Pro Leu Val Val Glu Ile Ser Glu

1 5 10 15

Glu Gly Glu Met Lys Ile Glu Glu Gly Lys Leu Val Ile Trp Ile Asn

20 25 30

Gly Asp Lys Gly Tyr Asn Gly Leu Ala Glu Val Gly Lys Lys Phe Glu

35 40 45

Lys Asp Thr Gly Ile Lys Val Thr Val Glu His Pro Asp Lys Leu Glu

50 55 60

Glu Lys Phe Pro Gln Val Ala Ala Thr Gly Asp Gly Pro Asp Ile Ile

65 70 75 80

Phe Trp Ala His Asp Arg Phe Gly Gly Tyr Ala Gln Ser Gly Leu Leu

85 90 95

Ala Glu Ile Thr Pro Asp Lys Ala Phe Gln Asp Lys Leu Tyr Pro Phe

100 105 110

Thr Trp Asp Ala Val Arg Tyr Asn Gly Lys Leu Ile Ala Tyr Pro Ile

115 120 125

Ala Val Glu Ala Leu Ser Leu Ile Tyr Asn Lys Asp Leu Leu Pro Asn

130 135 140

Pro Pro Lys Thr Trp Glu Glu Ile Pro Ala Leu Asp Lys Glu Leu Lys

145 150 155 160

Ala Lys Gly Lys Ser Ala Leu Met Phe Asn Leu Gln Glu Pro Tyr Phe

165 170 175

Thr Trp Pro Leu Ile Ala Ala Asp Gly Gly Tyr Ala Phe Lys Tyr Glu

180 185 190

Asn Gly Lys Tyr Asp Ile Lys Asp Val Gly Val Asp Asn Ala Gly Ala

195 200 205

Lys Ala Gly Leu Thr Phe Leu Val Asp Leu Ile Lys Asn Lys His Met

210 215 220

Asn Ala Asp Thr Asp Tyr Ser Ile Ala Glu Ala Ala Phe Asn Lys Gly

225 230 235 240

Glu Thr Ala Met Thr Ile Asn Gly Pro Trp Ala Trp Ser Asn Ile Asp

245 250 255

Thr Ser Lys Val Asn Tyr Gly Val Thr Val Leu Pro Thr Phe Lys Gly

260 265 270

Gln Pro Ser Lys Pro Phe Val Gly Val Leu Ser Ala Gly Ile Asn Ala

275 280 285

Ala Ser Pro Asn Lys Glu Leu Ala Lys Glu Phe Leu Glu Asn Tyr Leu

290 295 300

Leu Thr Asp Glu Gly Leu Glu Ala Val Asn Lys Asp Lys Pro Leu Gly

305 310 315 320

Ala Val Ala Leu Lys Ser Tyr Glu Glu Glu Leu Ala Lys Asp Pro Arg

325 330 335

Ile Ala Ala Thr Met Glu Asn Ala Gln Lys Gly Glu Ile Met Pro Asn

340 345 350

Ile Pro Gln Met Ser Ala Phe Trp Tyr Ala Val Arg Thr Ala Val Ile

355 360 365

Asn Ala Ala Ser Gly Arg Gln Thr Val Asp Glu Ala Leu Lys Asp Ala

370 375 380

Gln Thr Arg Ile Thr Lys Glu Asn Leu Tyr Phe Gln Gly Gly Gly Met

385 390 395 400

Val Lys His Trp Lys Asn Tyr Asn Cys Glu Gln His Val Val Tyr Thr

405 410 415

Lys Asp Asp Tyr Leu Leu Cys Ile His Arg Ile Pro Ser Val Lys Glu

420 425 430

Arg Asp Lys Lys Ala His Arg Tyr Glu Phe Lys Glu Glu Ile Glu Val

435 440 445

Ile Asp Asn Leu Asp Lys Phe Val Gln Thr Glu Thr Pro Lys Pro Leu

450 455 460

Gly Tyr Lys Gly Lys Pro Val Val Leu Leu Tyr His Gly Phe Leu Met

465 470 475 480

Ser Ser Glu Val Trp Val Ser Asn Thr Asp Glu Tyr Ser Asn Leu Pro

485 490 495

Phe Val Leu Ala Gln Arg Gly Tyr Asp Val Trp Leu Gly Asn Ala Arg

500 505 510

Gly Asn Lys Tyr Ser Gln Tyr His Leu His Leu Lys Leu Asp Asp Gln

515 520 525

Gln Phe Trp Asn Phe Ser Met Asn Glu Phe Val Met Arg Asp Leu Pro

530 535 540

Asp Thr Ile Asp Tyr Ile Leu Ala Gln Thr Gly Ala Pro Asn Leu Thr

545 550 555 560

Tyr Ile Gly Phe Ser Gln Gly Thr Ala Gln Ala Phe Ala Ser Leu Ser

565 570 575

Val Asn Pro Asp Leu Asn Lys Lys Ile Asn Leu Phe Ile Ala Leu Ala

580 585 590

Pro Ala Thr Thr Pro Lys Gly Leu Arg His Pro Ile Ile Asp Ala Phe

595 600 605

Val Lys Ala Thr Pro Thr Val Ile Tyr Leu Met Phe Gly Arg Lys Ala

610 615 620

Ala Leu Lys Leu Thr Leu Phe Trp Gln Arg Ile Val Ser Pro Pro Leu

625 630 635 640

Phe Thr Lys Ile Ile Asp Thr Cys Cys His Phe Leu Phe Gly Trp Thr

645 650 655

Gly Arg Asn Ile Ser Asp Ala Gln Lys Ala Val Ser Tyr Gln His Leu

660 665 670

Tyr Ser Ser Thr Ser Val Lys Ser Leu Val His Trp Phe Gln Ile Ile

675 680 685

Arg Cys Gly Gln Phe Gln Met Tyr Asp Glu Met Pro Ser Arg Leu Pro

690 695 700

Tyr His Thr Val Asn Ser Val Ala Asp His Val Pro Pro Arg Phe Pro

705 710 715 720

Thr Leu Gln Ile Thr Thr Pro Ile Ala Ile Phe Tyr Gly Arg Ser Asp

725 730 735

Ser Leu Val Asp Phe Asn Val Leu Ser Ala Asp Leu Pro Ser Pro Leu

740 745 750

Ala Tyr Val Lys Ser Ile Glu Lys Trp Glu His Leu Asp Phe Leu Trp

755 760 765

Ala Asp Gly Ile Asp Lys

770

<210> 12

<211> 855

<212> PRT

<213> 古菌甲基转移酶亚基A(MtrA)

<400> 12

Met Pro Gly Ala Phe Asp Ala Asp Pro Leu Val Val Glu Ile Ser Glu

1 5 10 15

Glu Gly Glu Met Gly His His His His His His Ser Pro Ile Leu Gly

20 25 30

Tyr Trp Lys Ile Lys Gly Leu Val Gln Pro Thr Arg Leu Leu Leu Glu

35 40 45

Tyr Leu Glu Glu Lys Tyr Glu Glu His Leu Tyr Glu Arg Asp Glu Gly

50 55 60

Asp Lys Trp Arg Asn Lys Lys Phe Glu Leu Gly Leu Glu Phe Pro Asn

65 70 75 80

Leu Pro Tyr Tyr Ile Asp Gly Asp Val Lys Leu Thr Gln Ser Met Ala

85 90 95

Ile Ile Arg Tyr Ile Ala Asp Lys His Asn Met Leu Gly Gly Cys Pro

100 105 110

Lys Glu Arg Ala Glu Ile Ser Met Leu Glu Gly Ala Val Leu Asp Ile

115 120 125

Arg Tyr Gly Val Ser Arg Ile Ala Tyr Ser Lys Asp Phe Glu Thr Leu

130 135 140

Lys Val Asp Phe Leu Ser Lys Leu Pro Glu Met Leu Lys Met Phe Glu

145 150 155 160

Asp Arg Leu Cys His Lys Thr Tyr Leu Asn Gly Asp His Val Thr His

165 170 175

Pro Asp Phe Met Leu Tyr Asp Ala Leu Asp Val Val Leu Tyr Met Asp

180 185 190

Pro Met Cys Leu Asp Ala Phe Pro Lys Leu Val Cys Phe Lys Lys Arg

195 200 205

Ile Glu Ala Ile Pro Gln Ile Asp Lys Tyr Leu Lys Ser Ser Lys Tyr

210 215 220

Ile Ala Trp Pro Leu Gln Gly Trp Gln Ala Thr Phe Gly Gly Gly Asp

225 230 235 240

His Pro Pro Lys Ser Asp Ser Gly Glu Asn Leu Tyr Phe Gln Gly Met

245 250 255

Pro Arg Pro Glu Leu Pro Leu Pro Glu Gly Trp Glu Glu Ala Arg Asp

260 265 270

Phe Asp Gly Lys Val Tyr Tyr Ile Asp His Thr Asn Arg Thr Thr Ser

275 280 285

Trp Ile Asp Pro Arg Asp Arg Tyr Thr Lys Pro Leu Thr Phe Ala Asp

290 295 300

Cys Ile Ser Asp Glu Leu Pro Leu Gly Trp Glu Glu Ala Tyr Asp Pro

305 310 315 320

Gln Val Gly Asp Tyr Phe Ile Asp His Asn Thr Lys Thr Thr Gln Ile

325 330 335

Glu Asp Pro Arg Val Gln Trp Arg Arg Glu Gln Glu His Met Leu Lys

340 345 350

Asp Tyr Leu Val Val Ala Gln Glu Ala Leu Ser Ala Gln Lys Glu Ile

355 360 365

Tyr Gln Val Lys Gln Gln Arg Leu Glu Leu Ala Gln Gln Glu Tyr Gln

370 375 380

Gln Leu His Ala Val Trp Glu His Lys Leu Gly Ser Gln Val Ser Leu

385 390 395 400

Val Ser Gly Ser Ser Ser Ser Ser Lys Tyr Asp Pro Glu Ile Leu Lys

405 410 415

Ala Glu Ile Ala Thr Ala Lys Ser Arg Val Asn Lys Leu Lys Arg Glu

420 425 430

Met Val His Leu Gln His Glu Leu Gln Phe Lys Glu Arg Gly Phe Gln

435 440 445

Thr Leu Lys Lys Ile Asp Lys Lys Met Ser Asp Ala Gln Gly Ser Tyr

450 455 460

Lys Leu Asp Glu Ala Gln Ala Val Leu Arg Glu Thr Lys Ala Ile Lys

465 470 475 480

Lys Ala Ile Thr Cys Gly Glu Lys Glu Lys Gln Asp Leu Ile Lys Ser

485 490 495

Leu Ala Met Leu Lys Asp Gly Phe Arg Thr Asp Arg Gly Ser His Ser

500 505 510

Asp Leu Trp Ser Ser Ser Ser Ser Leu Glu Ser Ser Ser Phe Pro Leu

515 520 525

Pro Lys Gln Tyr Leu Asp Val Ser Ser Gln Thr Asp Ile Ser Gly Ser

530 535 540

Phe Gly Ile Asn Ser Asn Asn Gln Leu Ala Glu Lys Val Arg Leu Arg

545 550 555 560

Leu Arg Tyr Glu Glu Ala Lys Arg Arg Ile Ala Asn Leu Lys Ile Gln

565 570 575

Leu Ala Lys Leu Asp Ser Glu Ala Trp Pro Gly Val Leu Asp Ser Glu

580 585 590

Arg Asp Arg Leu Ile Leu Ile Asn Glu Lys Glu Glu Leu Leu Lys Glu

595 600 605

Met Arg Phe Ile Ser Pro Arg Lys Trp Thr Gln Gly Glu Val Glu Gln

610 615 620

Leu Glu Met Ala Arg Lys Arg Leu Glu Lys Asp Leu Gln Ala Ala Arg

625 630 635 640

Asp Thr Gln Ser Lys Ala Leu Thr Glu Arg Leu Lys Leu Asn Ser Lys

645 650 655

Arg Asn Gln Leu Val Arg Glu Leu Glu Glu Ala Thr Arg Gln Val Ala

660 665 670

Thr Leu His Ser Gln Leu Lys Ser Leu Ser Ser Ser Met Gln Ser Leu

675 680 685

Ser Ser Gly Ser Ser Pro Gly Ser Leu Thr Ser Ser Arg Gly Ser Leu

690 695 700

Val Ala Ser Ser Leu Asp Ser Ser Thr Ser Ala Ser Phe Thr Asp Leu

705 710 715 720

Tyr Tyr Asp Pro Phe Glu Gln Leu Asp Ser Glu Leu Gln Ser Lys Val

725 730 735

Glu Phe Leu Leu Leu Glu Gly Ala Thr Gly Phe Arg Pro Ser Gly Cys

740 745 750

Ile Thr Thr Ile His Glu Asp Glu Val Ala Lys Thr Gln Lys Ala Glu

755 760 765

Gly Gly Gly Arg Leu Gln Ala Leu Arg Ser Leu Ser Gly Thr Pro Lys

770 775 780

Ser Met Thr Ser Leu Ser Pro Arg Ser Ser Leu Ser Ser Pro Ser Pro

785 790 795 800

Pro Cys Ser Pro Leu Met Ala Asp Pro Leu Leu Ala Gly Asp Ala Phe

805 810 815

Leu Asn Ser Leu Glu Phe Glu Asp Pro Glu Leu Ser Ala Thr Leu Cys

820 825 830

Glu Leu Ser Leu Gly Asn Ser Ala Gln Glu Arg Tyr Arg Leu Glu Glu

835 840 845

Pro Gly Thr Glu Gly Lys Gln

850 855

<210> 13

<211> 946

<212> PRT

<213> 古菌甲基转移酶亚基A(MtrA)

<400> 13

Met Pro Gly Ala Phe Asp Ala Asp Pro Leu Val Val Glu Ile Ser Glu

1 5 10 15

Glu Gly Glu Met Gly Ser Ser His His His His His His Glu Asn Leu

20 25 30

Tyr Phe Gln Gly Gly Gly Glu Arg Ala Met Glu Gln Leu Asn Arg Leu

35 40 45

Thr Arg Ser Leu Arg Arg Ala Arg Thr Val Glu Leu Pro Glu Asp Asn

50 55 60

Glu Thr Ala Val Tyr Thr Leu Met Pro Met Val Met Ala Asp Gln His

65 70 75 80

Arg Ser Val Ser Glu Leu Leu Ser Asn Ser Lys Phe Asp Val Asn Tyr

85 90 95

Ala Phe Gly Arg Val Lys Arg Ser Leu Leu His Ile Ala Ala Asn Cys

100 105 110

Gly Ser Val Glu Cys Leu Val Leu Leu Leu Lys Lys Gly Ala Asn Pro

115 120 125

Asn Tyr Gln Asp Ile Ser Gly Cys Thr Pro Leu His Leu Ala Ala Arg

130 135 140

Asn Gly Gln Lys Lys Cys Met Ser Lys Leu Leu Glu Tyr Ser Ala Asp

145 150 155 160

Val Asn Ile Cys Asn Asn Glu Gly Leu Thr Ala Ile His Trp Leu Ala

165 170 175

Val Asn Gly Arg Thr Glu Leu Leu His Asp Leu Val Gln His Val Ser

180 185 190

Asp Val Asp Val Glu Asp Ala Met Gly Gln Thr Ala Leu His Val Ala

195 200 205

Cys Gln Asn Gly His Lys Thr Thr Val Gln Cys Leu Leu Asp Ser Gly

210 215 220

Ala Asp Ile Asn Arg Pro Asn Val Ser Gly Ala Thr Pro Leu Tyr Phe

225 230 235 240

Ala Cys Ser His Gly Gln Arg Asp Thr Ala Gln Ile Leu Leu Leu Arg

245 250 255

Gly Ala Lys Tyr Leu Pro Asp Lys Asn Gly Val Thr Pro Leu Asp Leu

260 265 270

Cys Val Gln Gly Gly Tyr Gly Glu Thr Cys Glu Val Leu Ile Gln Tyr

275 280 285

His Pro Arg Leu Phe Gln Thr Ile Ile Gln Met Thr Gln Asn Glu Asp

290 295 300

Leu Arg Glu Asn Met Leu Arg Gln Val Leu Glu His Leu Ser Gln Gln

305 310 315 320

Ser Glu Ser Gln Tyr Leu Lys Ile Leu Thr Ser Leu Ala Glu Val Ala

325 330 335

Thr Thr Asn Gly His Lys Leu Leu Ser Leu Ser Ser Asn Tyr Asp Ala

340 345 350

Gln Met Lys Ser Leu Leu Arg Ile Val Arg Met Phe Cys His Val Phe

355 360 365

Arg Ile Gly Pro Ser Ser Pro Ser Asn Gly Ile Asp Met Gly Tyr Asn

370 375 380

Gly Asn Lys Thr Pro Arg Ser Gln Val Phe Lys Pro Leu Glu Leu Leu

385 390 395 400

Trp His Ser Leu Asp Glu Trp Leu Val Leu Ile Ala Thr Glu Leu Met

405 410 415

Lys Asn Lys Arg Asp Ser Thr Glu Ile Thr Ser Ile Leu Leu Lys Gln

420 425 430

Lys Gly Gln Asp Gln Asp Ala Ala Ser Ile Pro Pro Phe Glu Pro Pro

435 440 445

Gly Pro Gly Ser Tyr Glu Asn Leu Ser Thr Gly Thr Arg Glu Ser Lys

450 455 460

Pro Asp Ala Leu Ala Gly Arg Gln Glu Ala Ser Ala Asp Cys Gln Asp

465 470 475 480

Val Ile Ser Met Thr Ala Asn Arg Leu Ser Ala Val Ile Gln Ala Phe

485 490 495

Tyr Met Cys Cys Ser Cys Gln Met Pro Pro Gly Met Thr Ser Pro Arg

500 505 510

Phe Ile Glu Phe Val Cys Lys His Asp Glu Val Leu Lys Cys Phe Val

515 520 525

Asn Arg Asn Pro Lys Ile Ile Phe Asp His Phe His Phe Leu Leu Glu

530 535 540

Cys Pro Glu Leu Met Ser Arg Phe Met His Ile Ile Lys Ala Gln Pro

545 550 555 560

Phe Lys Asp Arg Cys Glu Trp Phe Tyr Glu His Leu His Ser Gly Gln

565 570 575

Pro Asp Ser Asp Met Val His Arg Pro Val Asn Glu Asn Asp Ile Leu

580 585 590

Leu Val His Arg Asp Ser Ile Phe Arg Ser Ser Cys Glu Val Val Ser

595 600 605

Lys Ala Asn Cys Ala Lys Leu Lys Gln Gly Ile Ala Val Arg Phe His

610 615 620

Gly Glu Glu Gly Met Gly Gln Gly Val Val Arg Glu Trp Phe Asp Ile

625 630 635 640

Leu Ser Asn Glu Ile Val Asn Pro Asp Tyr Ala Leu Phe Thr Gln Ser

645 650 655

Ala Asp Gly Thr Thr Phe Gln Pro Asn Ser Asn Ser Tyr Val Asn Pro

660 665 670

Asp His Leu Asn Tyr Phe Arg Phe Ala Gly Gln Ile Leu Gly Leu Ala

675 680 685

Leu Asn His Arg Gln Leu Val Asn Ile Tyr Phe Thr Arg Ser Phe Tyr

690 695 700

Lys His Ile Leu Gly Ile Pro Val Asn Tyr Gln Asp Val Ala Ser Ile

705 710 715 720

Asp Pro Glu Tyr Ala Lys Asn Leu Gln Trp Ile Leu Asp Asn Asp Ile

725 730 735

Ser Asp Leu Gly Leu Glu Leu Thr Phe Ser Val Glu Thr Asp Val Phe

740 745 750

Gly Ala Met Glu Glu Val Pro Leu Lys Pro Gly Gly Gly Ser Ile Leu

755 760 765

Val Thr Gln Asn Asn Lys Ala Glu Tyr Val Gln Leu Val Thr Glu Leu

770 775 780

Arg Met Thr Arg Ala Ile Gln Pro Gln Ile Asn Ala Phe Leu Gln Gly

785 790 795 800

Phe His Met Phe Ile Pro Pro Ser Leu Ile Gln Leu Phe Asp Glu Tyr

805 810 815

Glu Leu Glu Leu Leu Leu Ser Gly Met Pro Glu Ile Asp Val Ser Asp

820 825 830

Trp Ile Lys Asn Thr Glu Tyr Thr Ser Gly Tyr Glu Arg Glu Asp Pro

835 840 845

Val Ile Gln Trp Phe Trp Glu Val Val Glu Asp Ile Thr Gln Glu Glu

850 855 860

Arg Val Leu Leu Leu Gln Phe Val Thr Gly Ser Ser Arg Val Pro His

865 870 875 880

Gly Gly Phe Ala Asn Ile Met Gly Gly Ser Gly Leu Gln Asn Phe Thr

885 890 895

Ile Ala Ala Val Pro Tyr Thr Pro Asn Leu Leu Pro Thr Ser Ser Thr

900 905 910

Cys Ile Asn Met Leu Lys Leu Pro Glu Tyr Pro Ser Lys Glu Ile Leu

915 920 925

Lys Asp Arg Leu Leu Val Ala Leu His Cys Gly Ser Tyr Gly Tyr Thr

930 935 940

Met Ala

945

<210> 14

<211> 1170

<212> PRT

<213> 古菌甲基转移酶亚基A(MtrA)

<400> 14

Met Pro Gly Ala Phe Asp Ala Asp Pro Leu Val Val Glu Ile Ser Glu

1 5 10 15

Glu Gly Glu Met Gly Ser Ser His His His His His His Glu Asn Leu

20 25 30

Tyr Phe Gln Gly Gly Gly Pro Val Gln Ala Pro Gln Trp Thr Asp Phe

35 40 45

Leu Ser Cys Pro Ile Cys Thr Gln Thr Phe Asp Glu Thr Ile Arg Lys

50 55 60

Pro Ile Ser Leu Gly Cys Gly His Thr Val Cys Lys Met Cys Leu Asn

65 70 75 80

Lys Leu His Arg Lys Ala Cys Pro Phe Asp Gln Thr Thr Ile Asn Thr

85 90 95

Asp Ile Glu Leu Leu Pro Val Asn Ser Ala Leu Leu Gln Leu Val Gly

100 105 110

Ala Gln Val Pro Glu Gln Gln Pro Ile Thr Leu Cys Ser Gly Val Glu

115 120 125

Asp Thr Lys His Tyr Glu Glu Ala Lys Lys Cys Val Glu Glu Leu Ala

130 135 140

Leu Tyr Leu Lys Pro Leu Ser Ser Ala Arg Gly Val Gly Leu Asn Ser

145 150 155 160

Thr Thr Gln Ser Val Leu Ser Arg Pro Met Gln Arg Lys Leu Val Thr

165 170 175

Leu Val His Cys Gln Leu Val Glu Glu Glu Gly Arg Ile Arg Ala Met

180 185 190

Arg Ala Ala Arg Ser Leu Gly Glu Arg Thr Val Thr Glu Leu Ile Leu

195 200 205

Gln His Gln Asn Pro Gln Gln Leu Ser Ser Asn Leu Trp Ala Ala Val

210 215 220

Arg Ala Arg Gly Cys Gln Phe Leu Gly Pro Ala Met Gln Glu Glu Ala

225 230 235 240

Leu Lys Leu Val Leu Leu Ala Leu Glu Asp Gly Ser Ala Leu Ser Arg

245 250 255

Lys Val Leu Val Leu Phe Val Val Gln Arg Leu Glu Pro Arg Phe Pro

260 265 270

Gln Ala Ser Lys Thr Ser Ile Gly His Val Val Gln Leu Leu Tyr Arg

275 280 285

Ala Ser Cys Phe Lys Val Thr Lys Arg Asp Glu Asp Ser Ser Leu Met

290 295 300

Gln Leu Lys Glu Glu Phe Arg Thr Tyr Glu Ala Leu Arg Arg Glu His

305 310 315 320

Asp Ser Gln Ile Val Gln Ile Ala Met Glu Ala Gly Leu Arg Ile Ala

325 330 335

Pro Asp Gln Trp Ser Ser Leu Leu Tyr Gly Asp Gln Ser His Lys Ser

340 345 350

His Met Gln Ser Ile Ile Asp Lys Leu Gln Thr Pro Ala Ser Phe Ala

355 360 365

Gln Ser Val Gln Glu Leu Thr Ile Ala Leu Gln Arg Thr Gly Asp Pro

370 375 380

Ala Asn Leu Asn Arg Leu Arg Pro His Leu Glu Leu Leu Ala Asn Ile

385 390 395 400

Asp Pro Ser Pro Asp Ala Pro Pro Pro Thr Trp Glu Gln Leu Glu Asn

405 410 415

Gly Leu Val Ala Val Arg Thr Val Val His Gly Leu Val Asp Tyr Ile

420 425 430

Gln Asn His Ser Lys Lys Gly Ala Asp Gln Gln Gln Pro Pro Gln His

435 440 445

Ser Lys Tyr Lys Thr Tyr Met Cys Arg Asp Met Lys Gln Arg Gly Gly

450 455 460

Cys Pro Arg Gly Ala Ser Cys Thr Phe Ala His Ser Gln Glu Glu Leu

465 470 475 480

Glu Lys Phe Arg Lys Met Asn Lys Arg Leu Val Pro Arg Arg Pro Leu

485 490 495

Ser Ala Ser Leu Gly Gln Leu Asn Glu Val Gly Leu Pro Ser Ala Ala

500 505 510

Ile Leu Pro Asp Glu Gly Ala Val Asp Leu Pro Ser Arg Lys Pro Pro

515 520 525

Ala Leu Pro Asn Gly Ile Val Ser Thr Gly Asn Thr Val Thr Gln Leu

530 535 540

Ile Pro Arg Gly Thr Asp Pro Ser Tyr Asp Ser Ser Leu Lys Pro Gly

545 550 555 560

Lys Ile Asp His Leu Ser Ser Ser Ala Pro Gly Ser Pro Pro Asp Leu

565 570 575

Leu Glu Ser Val Pro Lys Ser Ile Ser Ala Leu Pro Val Asn Pro His

580 585 590

Ser Ile Pro Pro Arg Gly Pro Ala Asp Leu Pro Pro Met Pro Val Thr

595 600 605

Lys Pro Leu Gln Met Val Pro Arg Gly Ser Gln Leu Tyr Pro Ala Gln

610 615 620

Gln Thr Asp Val Tyr Tyr Gln Asp Pro Arg Gly Ala Ala Pro Pro Phe

625 630 635 640

Glu Pro Ala Pro Tyr Gln Gln Gly Met Tyr Tyr Thr Pro Pro Pro Gln

645 650 655

Cys Val Ser Arg Phe Val Arg Pro Pro Pro Ser Ala Pro Glu Pro Ala

660 665 670

Pro Pro Tyr Leu Asp His Tyr Pro Pro Tyr Leu Gln Glu Arg Val Val

675 680 685

Asn Ser Gln Tyr Gly Thr Gln Pro Gln Gln Tyr Pro Pro Ile Tyr Pro

690 695 700

Ser His Tyr Asp Gly Arg Arg Val Tyr Pro Ala Pro Ser Tyr Thr Arg

705 710 715 720

Glu Glu Ile Phe Arg Glu Ser Pro Ile Pro Ile Glu Ile Pro Pro Ala

725 730 735

Ala Val Pro Ser Tyr Val Pro Glu Ser Arg Glu Arg Tyr Gln Gln Ile

740 745 750

Glu Ser Tyr Tyr Pro Val Ala Pro His Pro Thr Gln Ile Arg Pro Ser

755 760 765

Tyr Leu Arg Glu Pro Pro Tyr Ser Arg Leu Pro Pro Pro Pro Gln Pro

770 775 780

His Pro Ser Leu Asp Glu Leu His Arg Arg Arg Lys Glu Ile Met Ala

785 790 795 800

Gln Leu Glu Glu Arg Lys Val Ile Ser Pro Pro Pro Phe Ala Pro Ser

805 810 815

Pro Thr Leu Pro Pro Thr Phe His Pro Glu Glu Phe Leu Asp Glu Asp

820 825 830

Leu Lys Val Ala Gly Lys Tyr Lys Gly Asn Asp Tyr Ser Gln Tyr Ser

835 840 845

Pro Trp Ser Cys Asp Thr Ile Gly Ser Tyr Ile Gly Thr Lys Asp Ala

850 855 860

Lys Pro Lys Asp Val Val Ala Ala Gly Ser Val Glu Met Met Asn Val

865 870 875 880

Glu Ser Lys Gly Met Arg Asp Gln Arg Leu Asp Leu Gln Arg Arg Ala

885 890 895

Ala Glu Thr Ser Asp Asp Asp Leu Ile Pro Phe Gly Asp Arg Pro Thr

900 905 910

Val Ser Arg Phe Gly Ala Ile Ser Arg Thr Ser Lys Thr Ile Tyr Gln

915 920 925

Gly Ala Gly Pro Met Gln Ala Met Ala Pro Gln Gly Ala Pro Thr Lys

930 935 940

Ser Ile Asn Ile Ser Asp Tyr Ser Pro Tyr Gly Thr His Gly Gly Trp

945 950 955 960

Gly Ala Ser Pro Tyr Ser Pro His Gln Asn Ile Pro Ser Gln Gly His

965 970 975

Phe Ser Glu Arg Glu Arg Ile Ser Met Ser Glu Val Ala Ser His Gly

980 985 990

Lys Pro Leu Pro Ser Ala Glu Arg Glu Gln Leu Arg Leu Glu Leu Gln

995 1000 1005

Gln Leu Asn His Gln Ile Ser Gln Gln Thr Gln Leu Arg Gly Leu Glu

1010 1015 1020

Ala Val Ser Asn Arg Leu Val Leu Gln Arg Glu Ala Asn Thr Leu Ala

1025 1030 1035 1040

Gly Gln Ser Gln Pro Pro Pro Pro Pro Pro Pro Lys Trp Pro Gly Met

1045 1050 1055

Ile Ser Ser Glu Gln Leu Ser Leu Glu Leu His Gln Val Glu Arg Glu

1060 1065 1070

Ile Gly Lys Arg Thr Arg Glu Leu Ser Met Glu Asn Gln Cys Ser Leu

1075 1080 1085

Asp Met Lys Ser Lys Leu Asn Thr Ser Lys Gln Ala Glu Asn Gly Gln

1090 1095 1100

Pro Glu Pro Gln Asn Lys Val Pro Ala Glu Asp Leu Thr Leu Thr Phe

1105 1110 1115 1120

Ser Asp Val Pro Asn Gly Ser Ala Leu Thr Gln Glu Asn Ile Ser Leu

1125 1130 1135

Leu Ser Asn Lys Thr Ser Ser Leu Asn Leu Ser Glu Asp Pro Glu Gly

1140 1145 1150

Gly Gly Asp Asn Asn Asp Ser Gln Arg Ser Gly Val Thr Pro Ser Ser

1155 1160 1165

Ala Pro

1170

Claims (8)

1.一种基于蛋白连接酶的高通量检测蛋白表达的方法，其特征在于，将待测蛋白、蛋白连接酶和荧光多肽底物在反应缓冲液中混合，利用蛋白连接酶连接携带识别序列的目的蛋白质和荧光多肽底物，获得待测蛋白表达检测结果；

其中，所述蛋白连接酶的氨基酸序列如SEQ ID NO.1所示；

所述荧光多肽底物的氨基酸序列为依次连接的荧光素FITC、氨基己酸Ahx和PGA基序组成的氨基酸序列，具体的，所述荧光多肽底物的氨基酸序列为FITC-Ahx-RELASKDPGAFDADPLVVEISEEGE。

2.根据权利要求1所述的方法，其特征在于，所述反应缓冲液为50mM HEPESpH7.0、150mM NaCl、50mM KCl、5mM TCEP,反应温度为37℃,反应时间为15min。

3.一种荧光多肽底物，其特征在于，所述荧光多肽底物的氨基酸序列为FITC-Ahx-RELASKDPGAFDADPLVVEISEEGE。

4.一种蛋白连接酶，其特征在于，所述蛋白连接酶的氨基酸序列如SEQ ID NO.1所示。

5.一种核苷酸序列，其特征在于，所述核苷酸序列为用于编码如权利要求4所述蛋白连接酶的氨基酸序列的核苷酸序列。

6.根据权利要求5所述的一种核苷酸序列，其特征在于，所述核苷酸序列如SEQ IDNO.2所示。

7.一种蛋白表达载体，其特征在于，所述表达载体通过在pET-28a载体上插入如权利要求6所述的核苷酸序列获得。

8.一种蛋白表达系统，其特征在于，为转入权利要求7所述蛋白表达载体的大肠杆菌BL21菌株。

Images