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CN113801866B - 一种高效表达具有高活性及稳定性的重组tev酶及其制备方法和应用 - Google Patents

一种高效表达具有高活性及稳定性的重组tev酶及其制备方法和应用 Download PDF

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CN113801866B
CN113801866B CN202111024156.XA CN202111024156A CN113801866B CN 113801866 B CN113801866 B CN 113801866B CN 202111024156 A CN202111024156 A CN 202111024156A CN 113801866 B CN113801866 B CN 113801866B
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王峰
黄亚亮
徐霞
欧阳莹
缪奇微
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Abstract

本发明公开了一种高效表达具有高活性及稳定性的重组TEV酶及其制备、测定方法和应用，包括编码TEV蛋白酶的氨基酸序列或其突变序列，所述编码TEV蛋白酶的氨基酸序列或其突变序列的N端融合有CL7标签，所述编码TEV蛋白酶的氨基酸序列或其突变序列的C端融合有多聚精氨酸标签。与现有重组TEV蛋白酶相比，本发明所提供的重组TEV蛋白酶不仅能够克服野生型TEV蛋白酶在表达过程中对自身特异位点进行剪切而导致的蛋白稳定性及活性降低的缺陷，还具备更高的蛋白产量，更好的蛋白稳定性以及更高的活性，更加适合大规模生产及工业使用。

Description

一种高效表达具有高活性及稳定性的重组TEV酶及其制备方 法和应用
技术领域
本发明涉及重组蛋白技术领域，具体涉及一种高效表达具有高活性及稳定性的重组TEV酶及其制备方法和应用。
背景技术
重组蛋白的表达被广泛应用于生物工程当中，包括蛋白纯化，定位及功能分析的等。根据表达系统的不同，主要分为大肠杆菌表达系统、酵母表达系统、昆虫细胞表达系统以及哺乳动物细胞表达系统。其中酵母表达系统、昆虫表达系统以及哺乳动物表达系统属于真核表达系统，大肠杆菌表达系统属于原核表达系统。原核表达系统相较于真核表达系统拥有诸多优势，包括遗传背景清晰、繁殖快、成本低、抗污染能力强、表达量高、商品化载体和菌株种类齐全、适用范围广等。但大肠杆菌表达系统由于缺少真核生物中翻译后修饰所需的酶类，并且蛋白表达量过高或合成速度过快，以至于没有足够的时间进行折叠，二硫键不能正确配对，导致蛋白常常以包涵体的形式存在。包涵体的表达虽然有利于目的蛋白的初步纯化，但无生物活性的不溶性蛋白要经过变性及复性，使其重新折叠成具有天然蛋白构象和良好的生物活性的蛋白质是非常困难的。
为了解决该问题，利用DNA体外重组技术，在目的蛋白N端或C端添加可融合表达的特定蛋白、多肽或寡肽标签。通过添加融合标签，既能保留天然蛋白的大部分结构，又能实现提高蛋白溶解度、稳定性、促进其分泌等目的，并且重组蛋白通过融合标签与包被在固相基质上的特异配基结合，可使重组蛋白定向固定得以纯化，大大简化了重组蛋白的纯化过程。然而，研究发现大多数融合标签对蛋白的生物功能及结构都存在潜在干扰，因此常常需要在融合标签及目的蛋白之间添加一段特异的蛋白酶识别序列，并在后续纯化过程中使用相应的蛋白酶将其切割分开，最终获得目的蛋白。目前常用的切割融合标签及目的蛋白的工具酶有凝血酶，肠激酶，因子Xa，然而这些哺乳动物类的蛋白酶没有严格的序列特异性，可能会切割目的蛋白的氨基酸序列。从而对一些病毒类来源的蛋白酶研究表明其具有更好的序列特异性。
TEV蛋白酶是来源于烟草蚀纹病毒(TEV)的Nla蛋白酶，具有很强的位点特异性，能够识别EXXYXQ(G/S)的七氨基酸序列，TEV酶具有广泛的pH和温度耐受性，pH耐受范围在4-8.0；耐受温度在4-34℃,因此可作为理想的工具酶使用(Sun C,Liang J,Shi R,etal.Tobacco etch virus protease retains its activity in various buffers and inthe presence of diverse additives.[J].Protein Expr Purif,2012,82(1):226-231.)。然而，野生型的TEV蛋白酶在大肠杆菌中表达及纯化中存在三个问题：自我剪切、稀有密码子以及可溶性差。野生型TEV酶在纯化及储存过程中，会在特定位点自我剪切，不仅大幅度降低了TEV酶的活性，并且由于TEV蛋白酶截短体的存在，使得后续纯化过程中很难将TEV蛋白酶截短体与目的蛋白分开，蛋白酶纯度得不到保障(Kapust R B,Tzsér József,Fox J D,et al.Tobacco etch virus protease:mechanism of autolysis and rationaldesign of stable mutants with wild-type catalytic proficiency[J].ProteinEngineering,2001(12):993.)。研究发现，将野生型TEV蛋白酶的219位的丝氨酸突变为缬氨酸或者天冬酰胺可以大大抑制TEV酶的自我剪切(Kapust R B,Tzsér József,Fox J D,et al.Tobacco etch virus protease:mechanism of autolysis and rational designof stable mutants with wild-type catalytic proficiency[J].ProteinEngineering,2001(12):993.)。Nam,H等人发现通过在目的蛋白的C端加上多聚精氨酸(RRRRR)多肽，可以提高TEV蛋白酶的可溶性。为了解决TEV酶在表达过程中出现的可溶性差蛋白产量低的问题(Nam H,Hwang B J,Choi D,et al.Tobacco etch virus(TEV)proteasewith multiple mutations to improve solubility and reduce self-cleavageexhibits enhanced enzymatic activity[J].FEBS Open Bio,2020(4):619-626.)，Blommel,P.G等人通过在N端添加融合MBP标签以及去除C端6个氨基酸序列，结合自动诱导培养的方法提高了蛋白的可溶性(Blommel P G,Fox B G.A combined approach toimproving large-scale production of tobacco etch virus protease.[J].ProteinExpression&Purification,2007,55(1):53-68.)。此外，FangJ等人还通过突变氨基酸的方式构建了三种突变体蛋白包括TEV酶双突变体(L56V/S135G)，三突变体(T17S/N68D/I77V)及五突变体(T17S/L56V/N68D/I77V/S135G)(Fang J,Chen L,Cheng B,et al.Engineeringsoluble tobacco etch virus protease accompanies the loss of stability[J].Protein Expression&Purification,2013,92(1):29-35.)。比较三种TEV蛋白酶突变体在不同温度、变性剂下及不同培养条件下蛋白的溶解度和稳定性发现五突变体表现出最高的溶解度和热稳定性。虽然双突变体对变性剂具有最强的耐受性，但是可溶性最差的野生型TEV蛋白酶对变性剂的耐受性优于三突变体和五突变体。总体来说，虽然已经有很多的研究通过在TEV蛋白序列的N端添加助融标签、设计氨基酸突变的方式提高了TEV蛋白酶的表达量，但是这些改造后的突变体蛋白仍然存在一些局限性，大部分突变体蛋白虽然提高了蛋白的表达量，但是酶的活性却有所下降。基于上述内容，提出一种高效表达具有高活性及稳定性的重组TEV酶及其制备方法和应用。
发明内容
本发明的目的在于克服现有技术的不足，提供了一种高效表达具有高活性及稳定性的重组TEV酶及其制备方法和应用。
本发明通过以下技术方案来实现上述目的：
本发明提供了一种高效表达具有高活性及稳定性的重组TEV酶，包括编码TEV蛋白酶的氨基酸序列或其突变序列，所述编码TEV蛋白酶的氨基酸序列或其突变序列的N端融合有CL7标签，所述编码TEV蛋白酶的氨基酸序列或其突变序列的C端融合有多聚精氨酸标签。
进一步改进在于，所述CL7标签为依次顺式连接的His6-CL7-GGS多肽序列，所述CL7的氨基酸序列如SEQ ID NO.1所示。
进一步改进在于，所述多聚精氨酸标签为RRRGRRRGRRRG多肽序列组成。
进一步改进在于，所述编码TEV蛋白酶的氨基酸序列的突变序列为TEV蛋白酶的氨基酸序列中包括如下a)-f)中至少一种或多种突变：
a)TEV蛋白酶的第219位氨基酸残基由丝氨酸突变为缬氨酸；
b)TEV蛋白酶的第17位氨基酸残基由苏氨酸突变为丝氨酸；
c)TEV蛋白酶的第56位氨基酸残基由亮氨酸突变为缬氨酸；
d)TEV蛋白酶的第68位氨基酸残基由天冬酰胺突变为天冬氨酸；
e)TEV蛋白酶的第77位氨基酸残基由异亮氨酸突变为缬氨酸；
f)TEV蛋白酶的第135位氨基酸残基由丝氨酸突变为甘氨酸。
进一步改进在于，所述编码TEV蛋白酶的氨基酸序列的突变序列为TEV蛋白酶的氨基酸序列中包括a)-f)六种突变。
进一步改进在于，所述编码TEV蛋白酶的氨基酸序列如SEQ ID NO.2所示。
本发明还提供了一种多核苷酸序列，所述多核苷酸序列为用于编码上述高效表达具有高活性及稳定性的重组TEV酶的氨基酸序列的核苷酸序列。
进一步改进在于，所述多核苷酸序列如SEQ ID NO.3所示。
本发明还提供了一种重组质粒，所述重组质粒为将上述多核苷酸序列插入到表达载体中，且能够翻译表达出上述的重组TEV酶的重组质粒。
进一步改进在于，所述表达载体为pET-28a载体。
本发明还提供了一种重组工程菌，所述重组工程菌包含上述重组质粒或基因组中整合有上述多核苷酸序列。
进一步改进在于，所述重组工程菌为大肠杆菌BL21(DE3)或T7 Express细胞。
本发明还提供了一种上述重组TEV酶的制备方法，步骤包括：
(1)以烟草蚀纹病毒TEV的基因序列为模板，通过PCR扩增技术获得TEV蛋白酶突变体的基因，将其基因构建在pET-28a载体上，获得重组质粒。
(2)利用大肠杆菌原核表达系统表达重组质粒，并利用Ni-NTA柱和亲和层析纯化获得重组TEV酶蛋白。
进一步改进在于，纯化重组TEV酶蛋白的缓冲液为50mMTris-HCl,500mMNaCl,10％glycerol,20mM imidazole。
本发明还提供了一种上述重组TEV酶活性测定方法，使用荧光共振能量转移方法检测所述TEV蛋白酶活性。
本发明还提供了一种上述重组TEV酶热稳定性测定方法，通过测定突变体蛋白溶解温度Tm值的变化判断蛋白稳定性，Tm值增大，蛋白越稳定。
本发明的有益效果在于：与现有重组TEV蛋白酶相比，本发明所提供的重组TEV蛋白酶不仅能够克服野生型TEV蛋白酶在表达过程中对自身特异位点进行剪切而导致的蛋白稳定性及活性降低的缺陷，还具备更高的蛋白产量，更好的蛋白稳定性以及更高的活性，更加适合大规模生产及工业使用。
本发明提供的重组TEV蛋白酶在TEV蛋白酶的N端和C端均融合了氨基酸多肽序列，其中的N端多肽序列为His-CL7-GGS，C端的多肽序列为RRRGRRRGRRRG。本发明提供的重组TEV蛋白酶对野生型TEV蛋白酶进行了突变，突变至少6个氨基酸残基。
本发明提供的重组TEV蛋白酶通过在N端添加CL7标签增加了TEV蛋白酶的表达量；通过在C端添加RRRGRRRGRRRG标签增加了TEV蛋白酶的溶解性；通过在N端和C端分别添加CL7和RRRGRRRGRRRG标签，优化突变氨基酸的数量增加了TEV蛋白酶的活性；通过在N端和C端分别添加CL7和RRRGRRRGRRRG标签，优化突变氨基酸的数量提高了TEV蛋白酶的Tm值、增加了蛋白的稳定性。
与现有TEV突变体(cTEV)相比，本发明提供的TEV蛋白酶突变体具有如下有益效果：
1、超高的蛋白产量。本发明提供的TEV蛋白酶突变体蛋白产量可高达61.95mg/L，并且蛋白纯度也可达到90％，而同等条件下，现有TEV蛋白酶突变体(cTEV)的产量仅为32.5mg/L。
2、较好的活性。本发明提供的TEV蛋白酶突变体活性测定为8.09×10^-3(RFU/s/nM)，而同等条件下，现有TEV蛋白酶突变体(cTEV)活性测定为4.55×10^-3(RFU/s/nM)。本发明所述提供的TEV蛋白酶突变体活性相比于TEV蛋白酶突变体(cTEV)高出近2倍。
3、更高的热稳定性。本发明提供的TEV蛋白酶突变体Tm值为55.8℃。而同等条件下，现有TEV蛋白酶突变体(cTEV)Tm值为42.05℃，本发明所述提供的TEV蛋白酶突变体活性相比于TEV蛋白酶突变体(cTEV)具有更好的蛋白热稳定性，能够在更宽的温度范围内保持良好蛋白酶活性。
附图说明
图1为cTEV、prTEV蛋白酶在T7 Express细胞中表达纯化结果；
图2为cTEV、prTEV蛋白酶在BL21细胞中表达纯化结果；
图3为prTEV蛋白酶的质谱和分子筛检测检测结果；
图4为不同TEV蛋白酶突变体活性检测结果；
图5为cTEV、prTEV蛋白酶热稳定性测定结果；
图6为4℃下cTEV、prTEV蛋白酶酶切效果验证结果；
图7为室温下cTEV、prTEV蛋白酶酶切效果验证结果。
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
本实施例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，所用的试剂等材料，如无特别说明，均为市售购买产品。
2、方法
2.1质粒构建及小试表达
本发明使用的所有质粒均使用常规的分子生物学手段进行引物设计，PCR及载体片段重组构建得到，所有重组质粒均经测序验证与目标序列完全一致。
本发明通过使用多种常规表达载体，不断优化N端及C端融合多肽序列，并设计了不同氨基酸序列突变体及其组合构建了多个重组质粒，最终得到了同时具备高产量，高稳定性及高活性三种特征的重组TEV蛋白酶。
本发明所述重组TEV蛋白酶的基因序列是通过常规PCR获得，烟草蚀纹病毒TEV的基因序列如SEQ ID NO.3所示，分别在N端和C端融合了多肽序列或标签，其中的N端多肽序列为His-CL7-GGS，CL7的氨基酸序列如SEQ ID NO.1所示，C端的多肽序列为RRRGRRRGRRRG，同时突变了如下6个氨基酸残基：第17位氨基酸残基由苏氨酸突变为丝氨酸；第56位氨基酸残基由亮氨酸突变为缬氨酸；第68位氨基酸残基由天冬酰胺突变为天冬氨酸；第77位氨基酸残基由异亮氨酸突变为缬氨酸；第219位氨基酸残基由缬氨酸突变为天冬酰胺；第135位氨基酸残基由丝氨酸突变为甘氨酸，重组TEV蛋白酶编号prTEV。
同时根据现有酶活性较高的突变体进行改造(Kapust R B,Tzsér József,Fox JD,et al.Tobacco etch virus protease:mechanism of autolysis and rationaldesign of stable mutants with wild-type catalytic proficiency[J].ProteinEngineering,2001(12):993.)，获得编号为cTEV的重组TEV蛋白酶，所述cTEV与本发明prTEV蛋白酶突变体不同的是cTEV仅突变一个位点，即将第219位氨基酸残基由丝氨酸突变为缬氨酸，N端标签为MBP-TEV-His，C端助溶标签为5R。
2.1.1prTEV、cTEV重组TEV蛋白酶质粒转化大肠杆菌感受态细胞
从-80℃冰箱取出感受态细胞于冰上化冻，超净台内将所述重组质粒使用常规分子生物学手段分别转化BL21(DE3)和T7 Express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，将感受态涂布在LB固体平板上，37℃倒置过夜培养。
2.1.2prTEV、cTEV重组TEV蛋白酶小量表达
挑取过夜培养的单克隆菌落至5mlLB液体培养基中，37℃培养，待菌液OD600至0.6-0.8时取少量菌液用loading buffer进行固定，并取少量菌液加入甘油冻至-80℃，剩余菌液加入0.5mM IPTG诱导4个小时后，收集菌体并取诱导后菌液进行SDS-PAGE检测。
2.2蛋白的大量表达及纯化
2.2.1 prTEV重组TEV蛋白酶大量表达
将小试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1L LB液体培养基中，37℃培养至菌液OD600为0.6-0.8时加入0.5mMIPTG16℃培养过夜，5000rpm离心收集菌体。
2.2.2 prTEV重组TEV蛋白酶纯化
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(pH8.0),500mM NaCl,10％glycerol,20mM imidazole)，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HisFF富集纯化蛋白，纯化前先用裂解buffer平衡HisFF柱，将所有细胞上清与HisFF结合后，用不同梯度的咪唑溶液进行洗脱，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和图2。根据纯化的结果可知prTEV突变体TEV蛋白酶在T7 Express和BL21细胞中比现有的cTEV蛋白酶的表达量要高。
2.2.3 prTEV重组TEV蛋白酶质量检测
将咪唑洗脱下的蛋白取少量分别进行质谱和分子筛检测。质谱检测结果显示prTEV重组TEV蛋白酶的分子量为44486Da与其理论分子量44458非常接近，表明HisFF纯化后的蛋白为目标蛋白，此外，分子筛的结果显示，prTEV重组TEV蛋白酶在溶液中以单体的形式存在，大大地提高了TEV蛋白酶实际应用中的有效酶量。实验结果见图3。
2.3活性测试
使用荧光共振能量转移方法检测所述TEV蛋白酶活性，即在两个不同的荧光基团中，一个荧光基团(供体Donor)的发射光谱与另一个基团(受体Acceptor)的吸收光谱有一定的重叠，并且两个荧光基团间的距离小于
Figure GDA0003605034110000081
会发生荧光能量由供体向受体转移，使得供体荧光强度比其单独存在是要低得多(荧光猝灭)。在含有特异性识别位点的多肽序列两端连接有FAM和TAMRA荧光基团，当多肽被剪切后，两个荧光基团分开，释放了强荧光信号，所述TEV蛋白酶活性越高，释放的荧光基团越多，荧光强度越大。酶活参数用每纳摩尔蛋白每秒钟内吸收的荧光强度表示。本发明所述TEV蛋白酶突变体相较于现有的TEV蛋白酶突变体具有更高的活性。
TEV蛋白酶活性测定方法具体操作如下：
将多肽底物5'-FAM-ENLYFQGSG-K(TAMRA)配制成1mM母液，分装备用。TEV蛋白酶测活缓冲液为50mM Tris-HCl(pH8.0),150mM NaCl,1mM DTT,0.5mM EDTA。用缓冲液将多肽浓度稀释成100nM，TEV蛋白酶从1μM以2倍梯度稀释，共稀释12个浓度。将30μL底物转移至384孔板中并设置两个复孔，转移30μL待测TEV蛋白酶至相应的孔板中，立即离心并振荡混匀，使用TECANF200酶标仪收集反应产生的荧光信号值。运用Graph Pad Prism9分析软件进行数据分析，最终获得待测蛋白酶的酶活参数。如图4即为Graph Pad Prism9软件分析获得不同TEV蛋白酶活参数。
2.4热稳定性测试
利用蛋白结构特点检测，蛋白具有疏水区结构并隐藏在内部，在温度上升时，蛋白的结构会被打开，暴露出疏水区，荧光染料SyproOrange就可以与该区域进行结合，并被激发荧光，根据荧光信号强度变化形成溶解曲线，溶解曲线导数最大值对应的温度即为熔点温度(Tm)。蛋白越稳定，测得的Tm值越大。
TEV蛋白酶热稳定性测定方法具体操作如下：
取5μgTEV蛋白酶加到96孔PCR板中，再加10×YPROOrange荧光染料至相应的孔中，将96孔PCR板放置qPCR仪中，设置仪器参数，按1分钟1℃的梯度从25℃升到99℃，计算蛋白的溶解曲线。如图5所示，两种蛋白酶热稳定性不同，所对应的Tm值也存在差异。
2.5进一步验证TEV蛋白酶突变体的酶切活性和稳定性
本实施例提供了一种TEV蛋白酶突变体，含有6个突变氨基酸残基，载体为pET28a，N端标签为8His-CL7-GGS，C端助溶标签为9R，命名为prTEV，氨基酸序列如SEQ ID NO：2所示。
为了研究本实施例中TEV蛋白酶突变体的酶切活性和稳定性，构建了一些对比例TEV蛋白酶突变体并将本实施例与对比例进行了比较。
其中对比例1为编号cTEV的重组TEV蛋白酶，载体为pET-28a，氨基酸序列如SEQ IDNO：4所示。
对比例2提供了一种TEV蛋白酶突变体，与本发明TEV蛋白酶突变体不同的是对比例仅突变一个位点，即将第219位氨基酸残基由丝氨酸突变为缬氨酸，载体为pET-28a，N端标签为MBP-TEV-His，C端助溶标签为9R，编号为rTEV1，氨基酸序列如SEQ ID NO：5所示。
对比例3提供了一种TEV蛋白酶突变体，与本发明TEV蛋白酶突变体不同的是对比例仅突变一个位点，即将第219位氨基酸残基由丝氨酸突变为缬氨酸，载体为pET28a，N端标签为His-GST-Thrombin，C端助溶标签为5R，编号为rTEV2，氨基酸序列如SEQ ID NO：6所示。
对比例4提供了一种TEV蛋白酶突变体，与本发明TEV蛋白酶突变体不同的是对比例仅突变一个位点，即将第219位氨基酸残基由丝氨酸突变为缬氨酸，载体为pET28a，N端标签为His，C端助溶标签为5R，编号为rTEV3，氨基酸序列如SEQ ID NO：7所示。
对比例5提供了一种TEV蛋白酶突变体，与本发明TEV蛋白酶突变体不同的是对比例仅突变一个位点，即将第219位氨基酸残基由丝氨酸突变为缬氨酸，载体为pET28a，N端标签为His-GST，C端助溶标签为5R，编号为rTEV4，氨基酸序列如SEQ ID NO：8所示。
对比例6提供了一种TEV蛋白酶突变体，与本发明TEV蛋白酶突变体不同的是对比例仅突变一个位点，即将第219位氨基酸残基由丝氨酸突变为缬氨酸，载体为pET28a，N端标签为His-CL7-GGS，C端助溶标签为5R，编号为rTEV5，氨基酸序列如SEQ ID NO：9所示。
对比例7提供了一种TEV蛋白酶突变体，与本发明TEV蛋白酶突变体不同的是其突变了两个位点，分别为第219位氨基酸残基由丝氨酸突变为缬氨酸，第153位氨基酸残基由丝氨酸突变为天冬酰胺，载体为pET28a，N端标签为His-CL7-GGS，C端助溶标签为9R，编号为rTEV6，氨基酸序列如SEQ ID NO：10所示。
对比例8提供了一种TEV蛋白酶突变体，与本发明TEV蛋白酶突变体不同的是对比例仅突变一个位点，即将第219位氨基酸残基由丝氨酸突变为缬氨酸，载体为pET28a，N端标签为His-CL7-GGS，C端助溶标签为9R，编号为rTEV7，氨基酸序列如SEQ ID NO：11所示。
对比例9提供了一种TEV蛋白酶突变体，与本发明TEV蛋白酶突变体同为六突变，不同之处是第219位氨基酸残基由丝氨酸突变为缬氨酸，载体为pET-28a，N端标签为MBP-TEV-His，C端助溶标签为5R，编号为rTEV8，氨基酸序列如SEQ ID NO：12所示。
表1 TEV蛋白酶突变体产量及热稳定性
Figure GDA0003605034110000111
表2 prTEV、cTEV、rTEV6、rTEV7突变位点比较
Figure GDA0003605034110000121
本实施例中提供的TEV蛋白酶突变体及所有对比例TEV蛋白酶突变体活性测定均在同等条件下获得。分别取相同浓度的蛋白与100nM5'-FAM-ENLYFQGSG-K(TAMRA)底物反应，使用TECANF200酶标仪监测TEV蛋白酶动力学活性。蛋白活性测定如图4所示，现有的TEV蛋白酶突变体cTEV与rTEV1蛋白酶突变体间的差异为C端助溶标签不同，结果表明C端标签为9R时，TEV蛋白酶突变体具有更高活性，故本发明优选地使用9R作为C端助溶标签。
本实施例中提供的TEV蛋白酶突变体及所有对比例TEV蛋白酶突变体产量标定均在同等条件下获得。分别取相同质量的蛋白与SYPROOrange荧光染料混匀，使用ABI7500qPCR仪，按照每分钟上升1℃的梯度从25℃升到99℃，获得溶解曲线计算蛋白Tm值。cTEV、rTEV2、rTEV3、rTEV4、rTEV5这5种TEV蛋白酶突变体的突变位点相同，C端标签均为5R，其差异为选用载体及融合标签不同。5种突变体蛋白活性测定结果如图4所示，5种TEV蛋白酶活性差异不大。各突变体的稳定性如表1所示，5种TEV蛋白酶Tm值差异不大，但从产量来看，最为突出的是rTEV5蛋白产量相较于其他高出2-4倍。故本发明优选地使用pET28a载体及His-CL7-GGS作为N端标签。
rTEV6、rTEV7及prTEV间的差异仅为突变氨基酸数目不同，即rTEV7为单突变TEV蛋白酶突变体(第219位氨基酸残基由丝氨酸突变为缬氨酸)，rTEV6为双突变TEV蛋白酶突变体(第219位氨基酸残基由丝氨酸突变为缬氨酸，第153位氨基酸残基由丝氨酸突变为天冬酰胺)，3种TEV蛋白酶突变体均使用pET28a载体，N端标签为His-CL7-GGS，C端助溶标签为9R。3种突变体蛋白活性测定结果如图4所示，3种突变体蛋白活性均高于现有的TEV蛋白酶突变体cTEV，其中本发明提供的六突变TEV蛋白酶prTEV活性同时高于二突变体TEV蛋白酶rTEV6和单突变体TEV蛋白酶rTEV7。对比三种突变体的Tm值，根据表1的结果可知，本发明提供的六突变TEV蛋白酶prTEV的Tm值远远高于单突变rTEV7和双突变rTEV6TEV蛋白酶，提示着prTEV具有更好的稳定性，可以在更高的温度下发挥活性。故本发明提供了一种高活性、高稳定性的TEV突变体蛋白酶。
prTEV与rTEV8均为六突变体且突变位点一致，其差异在于两者带有不同的载体和标签，本发明提供的TEV蛋白酶突变体优选地使用pET28a载体，N端以His-CL7-GGS为标签，C端添加了9R助溶标签，rTEV8使用pET28a为载体，MBP-TEV-His为N端标签，5R为C端助溶标签。2种突变体蛋白活性测定结果如图4所示，其中本发明提供的六突变TEV蛋白酶prTEV与rTEV8活性相近，但是根据表1的结果可知，本发明提供的六突变TEV蛋白酶prTEV产量比rTEV8高出3倍，Tm值高出5℃。因此认为prTEV具有更好的应用价值。
以上所述表明，本发明优选地，融合多肽标签及点突变的方法，可以在大肠杆菌异源表达系统中可以提高TEV蛋白酶的表达量和溶解性。同时这种融合标签和突变体的设计还可以大大提高TEV蛋白酶的活性和稳定性，从而使其具有更广阔的应用条件，具有更强的实际应用价值。
2.6酶切应用
为了测试prTEV的实际应用效果。用重组GST-TEV-Pro蛋白作为酶切对象，该重组蛋白N端带有TEV蛋白酶识别的TEV序列，因此TEV酶可以将该重组蛋白从TEV酶切位点切割成GST成Pro两个片段。
本实验中反应Buffer为50mMTris-HCl(pH8.0),500mMNaCl,5％glycerol。酶切反应分别在4℃和室温两种条件下进行测试。TEV蛋白酶与重组蛋白按照质量比为1:40进行孵育，分别收集1小时、2小时、4小时以及过夜不同时间点酶切后的样品进行SDS-PAGE检测，比较prTEV与cTEV的酶切效果。在4℃条件下，酶切1小时后，重组GST-TEV-Pro蛋白在两种TEV蛋白酶的作用下均有部分被切割成单独和GST和Pro片段，但是prTEV展现出更强的酶切效率。2小时、4小时，两种TEV蛋白酶的酶切效率差距更加明显。在过夜酶切后，两种TEV蛋白酶均基本将重组蛋白完全切开。实验结果见图6。
在常温下，prTEV和cTEV的酶切效果相差更加明显，在酶切2小时后，prTEV基本上将重组蛋白基本切开，而cTEV基本只切开了50％的重组蛋白。实验结果见图7。
以上所述表明，本发明优选地，融合多肽标签及点突变的方法，可以在大肠杆菌异源表达系统中可以提高TEV蛋白酶的表达量和溶解性。同时这种融合标签和突变体的设计还可以大大提高TEV蛋白酶在实际应用中的酶切效果，具有更强的实际应用价值。
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> 一种高效表达具有高活性及稳定性的重组TEV酶及其制备、测定方法和应用
<141> 2021-09-02
<160> 12
<170> SIPOSequenceListing 1.0
<210> 1
<211> 210
<212> PRT
<213> 烟草蚀刻病毒(Tobacco etch virus)
<400> 1
Met Ser Gly Arg Ser Val Arg Ala Glu Thr Arg Ser Arg Ala Lys Asp
1 5 10 15
Asp Ile Lys Arg Val Met Ala Ala Ile Glu Lys Val Arg Lys Trp Glu
20 25 30
Lys Lys Trp Val Thr Val Gly Asp Thr Ser Leu Arg Ile Tyr Lys Trp
35 40 45
Val Pro Val Thr Glu Pro Lys Val Asp Asp Lys Asn Lys Asn Lys Lys
50 55 60
Lys Gly Lys Asp Glu Lys Cys Gly Ser Glu Val Thr Thr Pro Glu Asn
65 70 75 80
Ser Ser Ser Pro Gly Met Met Asp Met His Asp Asp Asn Ser Asn Gln
85 90 95
Ser Ser Ile Ala Asp Ala Ser Pro Ile Lys Gln Glu Asn Ser Ser Asn
100 105 110
Ser Ser Pro Ala Pro Glu Pro Asn Ser Ala Val Pro Ser Asp Gly Thr
115 120 125
Glu Ala Lys Val Asp Glu Ala Gln Ala Asp Gly Lys Glu His Pro Gly
130 135 140
Ala Glu Asp Ala Ser Asp Glu Gln Asn Ser Gln Ser Ser Met Glu His
145 150 155 160
Ser Met Asn Ser Ser Glu Lys Val Asp Arg Gln Pro Ser Gly Asp Ser
165 170 175
Gly Leu Ala Ala Glu Thr Ser Ala Ile Ser Gln Asp Leu Glu Gly Val
180 185 190
Pro Pro Ser Lys Lys Met Lys Leu Glu Ala Ser Gln Gln Asn Ser Glu
195 200 205
Glu Met
210
<210> 2
<211> 390
<212> PRT
<213> 烟草蚀刻病毒(Tobacco etch virus)
<400> 2
Met His His His His His His His His Ser Lys Ser Asn Glu Pro Gly
1 5 10 15
Lys Ala Thr Gly Glu Gly Lys Pro Val Asn Asn Lys Trp Leu Asn Asn
20 25 30
Ala Gly Lys Asp Leu Gly Ser Pro Val Pro Asp Arg Ile Ala Asn Lys
35 40 45
Leu Arg Asp Lys Glu Phe Glu Ser Phe Asp Asp Phe Arg Glu Thr Phe
50 55 60
Trp Glu Glu Val Ser Lys Asp Pro Glu Leu Ser Lys Gln Phe Ser Arg
65 70 75 80
Asn Asn Asn Asp Arg Met Lys Val Gly Lys Ala Pro Lys Thr Arg Thr
85 90 95
Gln Asp Val Ser Gly Lys Arg Thr Ser Phe Glu Leu Asn His Gln Lys
100 105 110
Pro Ile Glu Gln Asn Gly Gly Val Tyr Asp Met Asp Asn Ile Ser Val
115 120 125
Val Thr Pro Lys Arg Asn Ile Asp Ile Glu Gly Gly Gly Ser Gly Glu
130 135 140
Ser Leu Phe Lys Gly Pro Arg Asp Tyr Asn Pro Ile Ser Ser Ser Ile
145 150 155 160
Cys His Leu Thr Asn Glu Ser Asp Gly His Thr Thr Ser Leu Tyr Gly
165 170 175
Ile Gly Phe Gly Pro Phe Ile Ile Thr Asn Lys His Leu Phe Arg Arg
180 185 190
Asn Asn Gly Thr Leu Val Val Gln Ser Leu His Gly Val Phe Lys Val
195 200 205
Lys Asp Thr Thr Thr Leu Gln Gln His Leu Val Asp Gly Arg Asp Met
210 215 220
Ile Ile Ile Arg Met Pro Lys Asp Phe Pro Pro Phe Pro Gln Lys Leu
225 230 235 240
Lys Phe Arg Glu Pro Gln Arg Glu Glu Arg Ile Cys Leu Val Thr Thr
245 250 255
Asn Phe Gln Thr Lys Ser Met Ser Ser Met Val Ser Asp Thr Ser Cys
260 265 270
Thr Phe Pro Ser Gly Asp Gly Ile Phe Trp Lys His Trp Ile Gln Thr
275 280 285
Lys Asp Gly Gln Cys Gly Ser Pro Leu Val Ser Thr Arg Asp Gly Phe
290 295 300
Ile Val Gly Ile His Ser Ala Ser Asn Phe Thr Asn Thr Asn Asn Tyr
305 310 315 320
Phe Thr Ser Val Pro Lys Asn Phe Met Glu Leu Leu Thr Asn Gln Glu
325 330 335
Ala Gln Gln Trp Val Ser Gly Trp Arg Leu Asn Ala Asp Ser Val Leu
340 345 350
Trp Gly Gly His Lys Val Phe Met Asn Lys Pro Glu Glu Pro Phe Gln
355 360 365
Pro Val Lys Glu Ala Thr Gln Leu Met Asn Arg Arg Arg Gly Arg Arg
370 375 380
Arg Gly Arg Arg Arg Gly
385 390
<210> 3
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<212> DNA
<213> 烟草蚀刻病毒(Tobacco etch virus)
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atgcatcatc atcatcacca tcatcatagt aaaagcaatg aaccgggcaa agcaaccggt 60
gaaggtaaac cggttaataa taaatggctg aataacgccg gcaaagatct gggtagtccg 120
gttccggatc gcattgccaa taaactgcgt gataaagagt tcgaatcatt cgatgacttc 180
cgtgaaacct tctgggaaga agttagtaaa gatccggaac tgagcaaaca gttcagtcgt 240
aataataatg atcgtatgaa ggttggtaag gcaccgaaaa ccagaaccca ggatgttagc 300
ggtaaacgca ccagcttcga actgaatcat cagaaaccga ttgaacagaa tggcggtgtg 360
tatgatatgg ataatattag tgttgtgacc ccgaaacgta atattgatat tgaaggcggc 420
ggcagtggag aaagcttgtt taaggggccg cgtgattaca acccgatatc gagcagcatt 480
tgtcatttga cgaatgaatc tgatgggcac acaacatcgt tgtatggtat tggatttggt 540
cccttcatca ttacaaacaa gcacttgttt agaagaaata atggaacact ggtggtccaa 600
tcactacatg gtgtattcaa ggtcaaggat accacgactt tgcaacaaca cctcgtggat 660
gggagggaca tgataattat tcgcatgcct aaggatttcc caccatttcc tcaaaagctg 720
aaatttagag agccacaaag ggaagagcgc atatgtcttg tgacaaccaa cttccaaact 780
aagagcatgt ctagcatggt gtcagacact agttgcacat tcccttcagg tgatggcata 840
ttctggaagc attggattca aaccaaggat gggcagtgtg gcagtccatt agtatcaact 900
agagatgggt tcattgttgg tatacactca gcatcgaatt tcaccaacac aaacaattat 960
ttcacaagcg tgccgaaaaa cttcatggaa ttgttgacaa atcaggaggc gcagcagtgg 1020
gttagtggtt ggcgattaaa tgctgactca gtattgtggg ggggccataa agttttcatg 1080
aataaacctg aagagccttt tcagccagtt aaggaagcga ctcaactcat gaatcgtcgt 1140
cgtggtcgtc gccgtggccg tcgtcgtgga taatga 1176
<210> 4
<211> 623
<212> PRT
<213> 烟草蚀刻病毒(Tobacco etch virus)
<400> 4
Met Gly Lys Ile Glu Glu Gly Lys Leu Val Ile Trp Ile Asn Gly Asp
1 5 10 15
Lys Gly Tyr Asn Gly Leu Ala Glu Val Gly Lys Lys Phe Glu Lys Asp
20 25 30
Thr Gly Ile Lys Val Thr Val Glu His Pro Asp Lys Leu Glu Glu Lys
35 40 45
Phe Pro Gln Val Ala Ala Thr Gly Asp Gly Pro Asp Ile Ile Phe Trp
50 55 60
Ala His Asp Arg Phe Gly Gly Tyr Ala Gln Ser Gly Leu Leu Ala Glu
65 70 75 80
Ile Thr Pro Asp Lys Ala Phe Gln Asp Lys Leu Tyr Pro Phe Thr Trp
85 90 95
Asp Ala Val Arg Tyr Asn Gly Lys Leu Ile Ala Tyr Pro Ile Ala Val
100 105 110
Glu Ala Leu Ser Leu Ile Tyr Asn Lys Asp Leu Leu Pro Asn Pro Pro
115 120 125
Lys Thr Trp Glu Glu Ile Pro Ala Leu Asp Lys Glu Leu Lys Ala Lys
130 135 140
Gly Lys Ser Ala Leu Met Phe Asn Leu Gln Glu Pro Tyr Phe Thr Trp
145 150 155 160
Pro Leu Ile Ala Ala Asp Gly Gly Tyr Ala Phe Lys Tyr Glu Asn Gly
165 170 175
Lys Tyr Asp Ile Lys Asp Val Gly Val Asp Asn Ala Gly Ala Lys Ala
180 185 190
Gly Leu Thr Phe Leu Val Asp Leu Ile Lys Asn Lys His Met Asn Ala
195 200 205
Asp Thr Asp Tyr Ser Ile Ala Glu Ala Ala Phe Asn Lys Gly Glu Thr
210 215 220
Ala Met Thr Ile Asn Gly Pro Trp Ala Trp Ser Asn Ile Asp Thr Ser
225 230 235 240
Lys Val Asn Tyr Gly Val Thr Val Leu Pro Thr Phe Lys Gly Gln Pro
245 250 255
Ser Lys Pro Phe Val Gly Val Leu Ser Ala Gly Ile Asn Ala Ala Ser
260 265 270
Pro Asn Lys Glu Leu Ala Lys Glu Phe Leu Glu Asn Tyr Leu Leu Thr
275 280 285
Asp Glu Gly Leu Glu Ala Val Asn Lys Asp Lys Pro Leu Gly Ala Val
290 295 300
Ala Leu Lys Ser Tyr Glu Glu Glu Leu Ala Lys Asp Pro Arg Ile Ala
305 310 315 320
Ala Thr Met Glu Asn Ala Gln Lys Gly Glu Ile Met Pro Asn Ile Pro
325 330 335
Gln Met Ser Ala Phe Trp Tyr Ala Val Arg Thr Ala Val Ile Asn Ala
340 345 350
Ala Ser Gly Arg Gln Thr Val Asp Glu Ala Leu Lys Asp Ala Gln Thr
355 360 365
Asn Ser Gly Ser Gly Gly Ser Gly His His His His His His Gly Glu
370 375 380
Ser Leu Phe Lys Gly Pro Arg Asp Tyr Asn Pro Ile Ser Ser Thr Ile
385 390 395 400
Cys His Leu Thr Asn Glu Ser Asp Gly His Thr Thr Ser Leu Tyr Gly
405 410 415
Ile Gly Phe Gly Pro Phe Ile Ile Thr Asn Lys His Leu Phe Arg Arg
420 425 430
Asn Asn Gly Thr Leu Leu Val Gln Ser Leu His Gly Val Phe Lys Val
435 440 445
Lys Asn Thr Thr Thr Leu Gln Gln His Leu Ile Asp Gly Arg Asp Met
450 455 460
Ile Ile Ile Arg Met Pro Lys Asp Phe Pro Pro Phe Pro Gln Lys Leu
465 470 475 480
Lys Phe Arg Glu Pro Gln Arg Glu Glu Arg Ile Cys Leu Val Thr Thr
485 490 495
Asn Phe Gln Thr Lys Ser Met Ser Ser Met Val Ser Asp Thr Ser Cys
500 505 510
Thr Phe Pro Ser Ser Asp Gly Ile Phe Trp Lys His Trp Ile Gln Thr
515 520 525
Lys Asp Gly Gln Cys Gly Ser Pro Leu Val Ser Thr Arg Asp Gly Phe
530 535 540
Ile Val Gly Ile His Ser Ala Ser Asn Phe Thr Asn Thr Asn Asn Tyr
545 550 555 560
Phe Thr Ser Val Pro Lys Asn Phe Met Glu Leu Leu Thr Asn Gln Glu
565 570 575
Ala Gln Gln Trp Val Ser Gly Trp Arg Leu Asn Ala Asp Ser Val Leu
580 585 590
Trp Gly Gly His Lys Val Phe Met Val Lys Pro Glu Glu Pro Phe Gln
595 600 605
Pro Val Lys Glu Ala Thr Gln Leu Met Asn Arg Arg Arg Arg Arg
610 615 620
<210> 5
<211> 630
<212> PRT
<213> 烟草蚀刻病毒(Tobacco etch virus)
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Met Gly Lys Ile Glu Glu Gly Lys Leu Val Ile Trp Ile Asn Gly Asp
1 5 10 15
Lys Gly Tyr Asn Gly Leu Ala Glu Val Gly Lys Lys Phe Glu Lys Asp
20 25 30
Thr Gly Ile Lys Val Thr Val Glu His Pro Asp Lys Leu Glu Glu Lys
35 40 45
Phe Pro Gln Val Ala Ala Thr Gly Asp Gly Pro Asp Ile Ile Phe Trp
50 55 60
Ala His Asp Arg Phe Gly Gly Tyr Ala Gln Ser Gly Leu Leu Ala Glu
65 70 75 80
Ile Thr Pro Asp Lys Ala Phe Gln Asp Lys Leu Tyr Pro Phe Thr Trp
85 90 95
Asp Ala Val Arg Tyr Asn Gly Lys Leu Ile Ala Tyr Pro Ile Ala Val
100 105 110
Glu Ala Leu Ser Leu Ile Tyr Asn Lys Asp Leu Leu Pro Asn Pro Pro
115 120 125
Lys Thr Trp Glu Glu Ile Pro Ala Leu Asp Lys Glu Leu Lys Ala Lys
130 135 140
Gly Lys Ser Ala Leu Met Phe Asn Leu Gln Glu Pro Tyr Phe Thr Trp
145 150 155 160
Pro Leu Ile Ala Ala Asp Gly Gly Tyr Ala Phe Lys Tyr Glu Asn Gly
165 170 175
Lys Tyr Asp Ile Lys Asp Val Gly Val Asp Asn Ala Gly Ala Lys Ala
180 185 190
Gly Leu Thr Phe Leu Val Asp Leu Ile Lys Asn Lys His Met Asn Ala
195 200 205
Asp Thr Asp Tyr Ser Ile Ala Glu Ala Ala Phe Asn Lys Gly Glu Thr
210 215 220
Ala Met Thr Ile Asn Gly Pro Trp Ala Trp Ser Asn Ile Asp Thr Ser
225 230 235 240
Lys Val Asn Tyr Gly Val Thr Val Leu Pro Thr Phe Lys Gly Gln Pro
245 250 255
Ser Lys Pro Phe Val Gly Val Leu Ser Ala Gly Ile Asn Ala Ala Ser
260 265 270
Pro Asn Lys Glu Leu Ala Lys Glu Phe Leu Glu Asn Tyr Leu Leu Thr
275 280 285
Asp Glu Gly Leu Glu Ala Val Asn Lys Asp Lys Pro Leu Gly Ala Val
290 295 300
Ala Leu Lys Ser Tyr Glu Glu Glu Leu Ala Lys Asp Pro Arg Ile Ala
305 310 315 320
Ala Thr Met Glu Asn Ala Gln Lys Gly Glu Ile Met Pro Asn Ile Pro
325 330 335
Gln Met Ser Ala Phe Trp Tyr Ala Val Arg Thr Ala Val Ile Asn Ala
340 345 350
Ala Ser Gly Arg Gln Thr Val Asp Glu Ala Leu Lys Asp Ala Gln Thr
355 360 365
Asn Ser Gly Ser Gly Gly Ser Gly His His His His His His Gly Glu
370 375 380
Ser Leu Phe Lys Gly Pro Arg Asp Tyr Asn Pro Ile Ser Ser Thr Ile
385 390 395 400
Cys His Leu Thr Asn Glu Ser Asp Gly His Thr Thr Ser Leu Tyr Gly
405 410 415
Ile Gly Phe Gly Pro Phe Ile Ile Thr Asn Lys His Leu Phe Arg Arg
420 425 430
Asn Asn Gly Thr Leu Leu Val Gln Ser Leu His Gly Val Phe Lys Val
435 440 445
Lys Asn Thr Thr Thr Leu Gln Gln His Leu Ile Asp Gly Arg Asp Met
450 455 460
Ile Ile Ile Arg Met Pro Lys Asp Phe Pro Pro Phe Pro Gln Lys Leu
465 470 475 480
Lys Phe Arg Glu Pro Gln Arg Glu Glu Arg Ile Cys Leu Val Thr Thr
485 490 495
Asn Phe Gln Thr Lys Ser Met Ser Ser Met Val Ser Asp Thr Ser Cys
500 505 510
Thr Phe Pro Ser Ser Asp Gly Ile Phe Trp Lys His Trp Ile Gln Thr
515 520 525
Lys Asp Gly Gln Cys Gly Ser Pro Leu Val Ser Thr Arg Asp Gly Phe
530 535 540
Ile Val Gly Ile His Ser Ala Ser Asn Phe Thr Asn Thr Asn Asn Tyr
545 550 555 560
Phe Thr Ser Val Pro Lys Asn Phe Met Glu Leu Leu Thr Asn Gln Glu
565 570 575
Ala Gln Gln Trp Val Ser Gly Trp Arg Leu Asn Ala Asp Ser Val Leu
580 585 590
Trp Gly Gly His Lys Val Phe Met Val Lys Pro Glu Glu Pro Phe Gln
595 600 605
Pro Val Lys Glu Ala Thr Gln Leu Met Asn Arg Arg Arg Gly Arg Arg
610 615 620
Arg Gly Arg Arg Arg Gly
625 630
<210> 6
<211> 477
<212> PRT
<213> 烟草蚀刻病毒(Tobacco etch virus)
<400> 6
Met Gly Ser Ser His His His His His His Met Ser Pro Ile Leu Gly
1 5 10 15
Tyr Trp Lys Ile Lys Gly Leu Val Gln Pro Thr Arg Leu Leu Leu Glu
20 25 30
Tyr Leu Glu Glu Lys Tyr Glu Glu His Leu Tyr Glu Arg Asp Glu Gly
35 40 45
Asp Lys Trp Arg Asn Lys Lys Phe Glu Leu Gly Leu Glu Phe Pro Asn
50 55 60
Leu Pro Tyr Tyr Ile Asp Gly Asp Val Lys Leu Thr Gln Ser Met Ala
65 70 75 80
Ile Ile Arg Tyr Ile Ala Asp Lys His Asn Met Leu Gly Gly Cys Pro
85 90 95
Lys Glu Arg Ala Glu Ile Ser Met Leu Glu Gly Ala Val Leu Asp Ile
100 105 110
Arg Tyr Gly Val Ser Arg Ile Ala Tyr Ser Lys Asp Phe Glu Thr Leu
115 120 125
Lys Val Asp Phe Leu Ser Lys Leu Pro Glu Met Leu Lys Met Phe Glu
130 135 140
Asp Arg Leu Cys His Lys Thr Tyr Leu Asn Gly Asp His Val Thr His
145 150 155 160
Pro Asp Phe Met Leu Tyr Asp Ala Leu Asp Val Val Leu Tyr Met Asp
165 170 175
Pro Met Cys Leu Asp Ala Phe Pro Lys Leu Val Cys Phe Lys Lys Arg
180 185 190
Ile Glu Ala Ile Pro Gln Ile Asp Lys Tyr Leu Lys Ser Ser Lys Tyr
195 200 205
Ile Ala Trp Pro Leu Gln Gly Trp Gln Ala Thr Phe Gly Gly Gly Asp
210 215 220
His Pro Pro Lys Ser Asp Leu Val Pro Arg Gly Ser Gly Glu Ser Leu
225 230 235 240
Phe Lys Gly Pro Arg Asp Tyr Asn Pro Ile Ser Ser Thr Ile Cys His
245 250 255
Leu Thr Asn Glu Ser Asp Gly His Thr Thr Ser Leu Tyr Gly Ile Gly
260 265 270
Phe Gly Pro Phe Ile Ile Thr Asn Lys His Leu Phe Arg Arg Asn Asn
275 280 285
Gly Thr Leu Leu Val Gln Ser Leu His Gly Val Phe Lys Val Lys Asn
290 295 300
Thr Thr Thr Leu Gln Gln His Leu Ile Asp Gly Arg Asp Met Ile Ile
305 310 315 320
Ile Arg Met Pro Lys Asp Phe Pro Pro Phe Pro Gln Lys Leu Lys Phe
325 330 335
Arg Glu Pro Gln Arg Glu Glu Arg Ile Cys Leu Val Thr Thr Asn Phe
340 345 350
Gln Thr Lys Ser Met Ser Ser Met Val Ser Asp Thr Ser Cys Thr Phe
355 360 365
Pro Ser Ser Asp Gly Ile Phe Trp Lys His Trp Ile Gln Thr Lys Asp
370 375 380
Gly Gln Cys Gly Ser Pro Leu Val Ser Thr Arg Asp Gly Phe Ile Val
385 390 395 400
Gly Ile His Ser Ala Ser Asn Phe Thr Asn Thr Asn Asn Tyr Phe Thr
405 410 415
Ser Val Pro Lys Asn Phe Met Glu Leu Leu Thr Asn Gln Glu Ala Gln
420 425 430
Gln Trp Val Ser Gly Trp Arg Leu Asn Ala Asp Ser Val Leu Trp Gly
435 440 445
Gly His Lys Val Phe Met Val Lys Pro Glu Glu Pro Phe Gln Pro Val
450 455 460
Lys Glu Ala Thr Gln Leu Met Asn Arg Arg Arg Arg Arg
465 470 475
<210> 7
<211> 251
<212> PRT
<213> 烟草蚀刻病毒(Tobacco etch virus)
<400> 7
Met Gly Ser Ser His His His His His His Gly Glu Ser Leu Phe Lys
1 5 10 15
Gly Pro Arg Asp Tyr Asn Pro Ile Ser Ser Thr Ile Cys His Leu Thr
20 25 30
Asn Glu Ser Asp Gly His Thr Thr Ser Leu Tyr Gly Ile Gly Phe Gly
35 40 45
Pro Phe Ile Ile Thr Asn Lys His Leu Phe Arg Arg Asn Asn Gly Thr
50 55 60
Leu Leu Val Gln Ser Leu His Gly Val Phe Lys Val Lys Asn Thr Thr
65 70 75 80
Thr Leu Gln Gln His Leu Ile Asp Gly Arg Asp Met Ile Ile Ile Arg
85 90 95
Met Pro Lys Asp Phe Pro Pro Phe Pro Gln Lys Leu Lys Phe Arg Glu
100 105 110
Pro Gln Arg Glu Glu Arg Ile Cys Leu Val Thr Thr Asn Phe Gln Thr
115 120 125
Lys Ser Met Ser Ser Met Val Ser Asp Thr Ser Cys Thr Phe Pro Ser
130 135 140
Ser Asp Gly Ile Phe Trp Lys His Trp Ile Gln Thr Lys Asp Gly Gln
145 150 155 160
Cys Gly Ser Pro Leu Val Ser Thr Arg Asp Gly Phe Ile Val Gly Ile
165 170 175
His Ser Ala Ser Asn Phe Thr Asn Thr Asn Asn Tyr Phe Thr Ser Val
180 185 190
Pro Lys Asn Phe Met Glu Leu Leu Thr Asn Gln Glu Ala Gln Gln Trp
195 200 205
Val Ser Gly Trp Arg Leu Asn Ala Asp Ser Val Leu Trp Gly Gly His
210 215 220
Lys Val Phe Met Val Lys Pro Glu Glu Pro Phe Gln Pro Val Lys Glu
225 230 235 240
Ala Thr Gln Leu Met Asn Arg Arg Arg Arg Arg
245 250
<210> 8
<211> 468
<212> PRT
<213> 烟草蚀刻病毒(Tobacco etch virus)
<400> 8
Met Gly His His His His His His Ser Pro Ile Leu Gly Tyr Trp Lys
1 5 10 15
Ile Lys Gly Leu Val Gln Pro Thr Arg Leu Leu Leu Glu Tyr Leu Glu
20 25 30
Glu Lys Tyr Glu Glu His Leu Tyr Glu Arg Asp Glu Gly Asp Lys Trp
35 40 45
Arg Asn Lys Lys Phe Glu Leu Gly Leu Glu Phe Pro Asn Leu Pro Tyr
50 55 60
Tyr Ile Asp Gly Asp Val Lys Leu Thr Gln Ser Met Ala Ile Ile Arg
65 70 75 80
Tyr Ile Ala Asp Lys His Asn Met Leu Gly Gly Cys Pro Lys Glu Arg
85 90 95
Ala Glu Ile Ser Met Leu Glu Gly Ala Val Leu Asp Ile Arg Tyr Gly
100 105 110
Val Ser Arg Ile Ala Tyr Ser Lys Asp Phe Glu Thr Leu Lys Val Asp
115 120 125
Phe Leu Ser Lys Leu Pro Glu Met Leu Lys Met Phe Glu Asp Arg Leu
130 135 140
Cys His Lys Thr Tyr Leu Asn Gly Asp His Val Thr His Pro Asp Phe
145 150 155 160
Met Leu Tyr Asp Ala Leu Asp Val Val Leu Tyr Met Asp Pro Met Cys
165 170 175
Leu Asp Ala Phe Pro Lys Leu Val Cys Phe Lys Lys Arg Ile Glu Ala
180 185 190
Ile Pro Gln Ile Asp Lys Tyr Leu Lys Ser Ser Lys Tyr Ile Ala Trp
195 200 205
Pro Leu Gln Gly Trp Gln Ala Thr Phe Gly Gly Gly Asp His Pro Pro
210 215 220
Lys Ser Asp Gly Glu Ser Leu Phe Lys Gly Pro Arg Asp Tyr Asn Pro
225 230 235 240
Ile Ser Ser Thr Ile Cys His Leu Thr Asn Glu Ser Asp Gly His Thr
245 250 255
Thr Ser Leu Tyr Gly Ile Gly Phe Gly Pro Phe Ile Ile Thr Asn Lys
260 265 270
His Leu Phe Arg Arg Asn Asn Gly Thr Leu Leu Val Gln Ser Leu His
275 280 285
Gly Val Phe Lys Val Lys Asn Thr Thr Thr Leu Gln Gln His Leu Ile
290 295 300
Asp Gly Arg Asp Met Ile Ile Ile Arg Met Pro Lys Asp Phe Pro Pro
305 310 315 320
Phe Pro Gln Lys Leu Lys Phe Arg Glu Pro Gln Arg Glu Glu Arg Ile
325 330 335
Cys Leu Val Thr Thr Asn Phe Gln Thr Lys Ser Met Ser Ser Met Val
340 345 350
Ser Asp Thr Ser Cys Thr Phe Pro Ser Ser Asp Gly Ile Phe Trp Lys
355 360 365
His Trp Ile Gln Thr Lys Asp Gly Gln Cys Gly Ser Pro Leu Val Ser
370 375 380
Thr Arg Asp Gly Phe Ile Val Gly Ile His Ser Ala Ser Asn Phe Thr
385 390 395 400
Asn Thr Asn Asn Tyr Phe Thr Ser Val Pro Lys Asn Phe Met Glu Leu
405 410 415
Leu Thr Asn Gln Glu Ala Gln Gln Trp Val Ser Gly Trp Arg Leu Asn
420 425 430
Ala Asp Ser Val Leu Trp Gly Gly His Lys Val Phe Met Val Lys Pro
435 440 445
Glu Glu Pro Phe Gln Pro Val Lys Glu Ala Thr Gln Leu Met Asn Arg
450 455 460
Arg Arg Arg Arg
465
<210> 9
<211> 381
<212> PRT
<213> 烟草蚀刻病毒(Tobacco etch virus)
<400> 9
Met His His His His His His Ser Lys Ser Asn Glu Pro Gly Lys Ala
1 5 10 15
Thr Gly Glu Gly Lys Pro Val Asn Asn Lys Trp Leu Asn Asn Ala Gly
20 25 30
Lys Asp Leu Gly Ser Pro Val Pro Asp Arg Ile Ala Asn Lys Leu Arg
35 40 45
Asp Lys Glu Phe Glu Ser Phe Asp Asp Phe Arg Glu Thr Phe Trp Glu
50 55 60
Glu Val Ser Lys Asp Pro Glu Leu Ser Lys Gln Phe Ser Arg Asn Asn
65 70 75 80
Asn Asp Arg Met Lys Val Gly Lys Ala Pro Lys Thr Arg Thr Gln Asp
85 90 95
Val Ser Gly Lys Arg Thr Ser Phe Glu Leu Asn His Gln Lys Pro Ile
100 105 110
Glu Gln Asn Gly Gly Val Tyr Asp Met Asp Asn Ile Ser Val Val Thr
115 120 125
Pro Lys Arg Asn Ile Asp Ile Glu Gly Gly Gly Ser Gly Glu Ser Leu
130 135 140
Phe Lys Gly Pro Arg Asp Tyr Asn Pro Ile Ser Ser Thr Ile Cys His
145 150 155 160
Leu Thr Asn Glu Ser Asp Gly His Thr Thr Ser Leu Tyr Gly Ile Gly
165 170 175
Phe Gly Pro Phe Ile Ile Thr Asn Lys His Leu Phe Arg Arg Asn Asn
180 185 190
Gly Thr Leu Leu Val Gln Ser Leu His Gly Val Phe Lys Val Lys Asn
195 200 205
Thr Thr Thr Leu Gln Gln His Leu Ile Asp Gly Arg Asp Met Ile Ile
210 215 220
Ile Arg Met Pro Lys Asp Phe Pro Pro Phe Pro Gln Lys Leu Lys Phe
225 230 235 240
Arg Glu Pro Gln Arg Glu Glu Arg Ile Cys Leu Val Thr Thr Asn Phe
245 250 255
Gln Thr Lys Ser Met Ser Ser Met Val Ser Asp Thr Ser Cys Thr Phe
260 265 270
Pro Ser Ser Asp Gly Ile Phe Trp Lys His Trp Ile Gln Thr Lys Asp
275 280 285
Gly Gln Cys Gly Ser Pro Leu Val Ser Thr Arg Asp Gly Phe Ile Val
290 295 300
Gly Ile His Ser Ala Ser Asn Phe Thr Asn Thr Asn Asn Tyr Phe Thr
305 310 315 320
Ser Val Pro Lys Asn Phe Met Glu Leu Leu Thr Asn Gln Glu Ala Gln
325 330 335
Gln Trp Val Ser Gly Trp Arg Leu Asn Ala Asp Ser Val Leu Trp Gly
340 345 350
Gly His Lys Val Phe Met Val Lys Pro Glu Glu Pro Phe Gln Pro Val
355 360 365
Lys Glu Ala Thr Gln Leu Met Asn Arg Arg Arg Arg Arg
370 375 380
<210> 10
<211> 388
<212> PRT
<213> 烟草蚀刻病毒(Tobacco etch virus)
<400> 10
Met His His His His His His Ser Lys Ser Asn Glu Pro Gly Lys Ala
1 5 10 15
Thr Gly Glu Gly Lys Pro Val Asn Asn Lys Trp Leu Asn Asn Ala Gly
20 25 30
Lys Asp Leu Gly Ser Pro Val Pro Asp Arg Ile Ala Asn Lys Leu Arg
35 40 45
Asp Lys Glu Phe Glu Ser Phe Asp Asp Phe Arg Glu Thr Phe Trp Glu
50 55 60
Glu Val Ser Lys Asp Pro Glu Leu Ser Lys Gln Phe Ser Arg Asn Asn
65 70 75 80
Asn Asp Arg Met Lys Val Gly Lys Ala Pro Lys Thr Arg Thr Gln Asp
85 90 95
Val Ser Gly Lys Arg Thr Ser Phe Glu Leu Asn His Gln Lys Pro Ile
100 105 110
Glu Gln Asn Gly Gly Val Tyr Asp Met Asp Asn Ile Ser Val Val Thr
115 120 125
Pro Lys Arg Asn Ile Asp Ile Glu Gly Gly Gly Ser Gly Glu Ser Leu
130 135 140
Phe Lys Gly Pro Arg Asp Tyr Asn Pro Ile Ser Ser Thr Ile Cys His
145 150 155 160
Leu Thr Asn Glu Ser Asp Gly His Thr Thr Ser Leu Tyr Gly Ile Gly
165 170 175
Phe Gly Pro Phe Ile Ile Thr Asn Lys His Leu Phe Arg Arg Asn Asn
180 185 190
Gly Thr Leu Leu Val Gln Ser Leu His Gly Val Phe Lys Val Lys Asn
195 200 205
Thr Thr Thr Leu Gln Gln His Leu Ile Asp Gly Arg Asp Met Ile Ile
210 215 220
Ile Arg Met Pro Lys Asp Phe Pro Pro Phe Pro Gln Lys Leu Lys Phe
225 230 235 240
Arg Glu Pro Gln Arg Glu Glu Arg Ile Cys Leu Val Thr Thr Asn Phe
245 250 255
Gln Thr Lys Ser Met Ser Ser Met Val Ser Asp Thr Ser Cys Thr Phe
260 265 270
Pro Ser Ser Asp Gly Ile Phe Trp Lys His Trp Ile Gln Thr Lys Asp
275 280 285
Gly Gln Cys Gly Asn Pro Leu Val Ser Thr Arg Asp Gly Phe Ile Val
290 295 300
Gly Ile His Ser Ala Ser Asn Phe Thr Asn Thr Asn Asn Tyr Phe Thr
305 310 315 320
Ser Val Pro Lys Asn Phe Met Glu Leu Leu Thr Asn Gln Glu Ala Gln
325 330 335
Gln Trp Val Ser Gly Trp Arg Leu Asn Ala Asp Ser Val Leu Trp Gly
340 345 350
Gly His Lys Val Phe Met Val Lys Pro Glu Glu Pro Phe Gln Pro Val
355 360 365
Lys Glu Ala Thr Gln Leu Met Asn Arg Arg Arg Gly Arg Arg Arg Gly
370 375 380
Arg Arg Arg Gly
385
<210> 11
<211> 388
<212> PRT
<213> 烟草蚀刻病毒(Tobacco etch virus)
<400> 11
Met His His His His His His Ser Lys Ser Asn Glu Pro Gly Lys Ala
1 5 10 15
Thr Gly Glu Gly Lys Pro Val Asn Asn Lys Trp Leu Asn Asn Ala Gly
20 25 30
Lys Asp Leu Gly Ser Pro Val Pro Asp Arg Ile Ala Asn Lys Leu Arg
35 40 45
Asp Lys Glu Phe Glu Ser Phe Asp Asp Phe Arg Glu Thr Phe Trp Glu
50 55 60
Glu Val Ser Lys Asp Pro Glu Leu Ser Lys Gln Phe Ser Arg Asn Asn
65 70 75 80
Asn Asp Arg Met Lys Val Gly Lys Ala Pro Lys Thr Arg Thr Gln Asp
85 90 95
Val Ser Gly Lys Arg Thr Ser Phe Glu Leu Asn His Gln Lys Pro Ile
100 105 110
Glu Gln Asn Gly Gly Val Tyr Asp Met Asp Asn Ile Ser Val Val Thr
115 120 125
Pro Lys Arg Asn Ile Asp Ile Glu Gly Gly Gly Ser Gly Glu Ser Leu
130 135 140
Phe Lys Gly Pro Arg Asp Tyr Asn Pro Ile Ser Ser Thr Ile Cys His
145 150 155 160
Leu Thr Asn Glu Ser Asp Gly His Thr Thr Ser Leu Tyr Gly Ile Gly
165 170 175
Phe Gly Pro Phe Ile Ile Thr Asn Lys His Leu Phe Arg Arg Asn Asn
180 185 190
Gly Thr Leu Leu Val Gln Ser Leu His Gly Val Phe Lys Val Lys Asn
195 200 205
Thr Thr Thr Leu Gln Gln His Leu Ile Asp Gly Arg Asp Met Ile Ile
210 215 220
Ile Arg Met Pro Lys Asp Phe Pro Pro Phe Pro Gln Lys Leu Lys Phe
225 230 235 240
Arg Glu Pro Gln Arg Glu Glu Arg Ile Cys Leu Val Thr Thr Asn Phe
245 250 255
Gln Thr Lys Ser Met Ser Ser Met Val Ser Asp Thr Ser Cys Thr Phe
260 265 270
Pro Ser Ser Asp Gly Ile Phe Trp Lys His Trp Ile Gln Thr Lys Asp
275 280 285
Gly Gln Cys Gly Ser Pro Leu Val Ser Thr Arg Asp Gly Phe Ile Val
290 295 300
Gly Ile His Ser Ala Ser Asn Phe Thr Asn Thr Asn Asn Tyr Phe Thr
305 310 315 320
Ser Val Pro Lys Asn Phe Met Glu Leu Leu Thr Asn Gln Glu Ala Gln
325 330 335
Gln Trp Val Ser Gly Trp Arg Leu Asn Ala Asp Ser Val Leu Trp Gly
340 345 350
Gly His Lys Val Phe Met Val Lys Pro Glu Glu Pro Phe Gln Pro Val
355 360 365
Lys Glu Ala Thr Gln Leu Met Asn Arg Arg Arg Gly Arg Arg Arg Gly
370 375 380
Arg Arg Arg Gly
385
<210> 12
<211> 617
<212> PRT
<213> 烟草蚀刻病毒(Tobacco etch virus)
<400> 12
Met Gly Lys Ile Glu Glu Gly Lys Leu Val Ile Trp Ile Asn Gly Asp
1 5 10 15
Lys Gly Tyr Asn Gly Leu Ala Glu Val Gly Lys Lys Phe Glu Lys Asp
20 25 30
Thr Gly Ile Lys Val Thr Val Glu His Pro Asp Lys Leu Glu Glu Lys
35 40 45
Phe Pro Gln Val Ala Ala Thr Gly Asp Gly Pro Asp Ile Ile Phe Trp
50 55 60
Ala His Asp Arg Phe Gly Gly Tyr Ala Gln Ser Gly Leu Leu Ala Glu
65 70 75 80
Ile Thr Pro Asp Lys Ala Phe Gln Asp Lys Leu Tyr Pro Phe Thr Trp
85 90 95
Asp Ala Val Arg Tyr Asn Gly Lys Leu Ile Ala Tyr Pro Ile Ala Val
100 105 110
Glu Ala Leu Ser Leu Ile Tyr Asn Lys Asp Leu Leu Pro Asn Pro Pro
115 120 125
Lys Thr Trp Glu Glu Ile Pro Ala Leu Asp Lys Glu Leu Lys Ala Lys
130 135 140
Gly Lys Ser Ala Leu Met Phe Asn Leu Gln Glu Pro Tyr Phe Thr Trp
145 150 155 160
Pro Leu Ile Ala Ala Asp Gly Gly Tyr Ala Phe Lys Tyr Glu Asn Gly
165 170 175
Lys Tyr Asp Ile Lys Asp Val Gly Val Asp Asn Ala Gly Ala Lys Ala
180 185 190
Gly Leu Thr Phe Leu Val Asp Leu Ile Lys Asn Lys His Met Asn Ala
195 200 205
Asp Thr Asp Tyr Ser Ile Ala Glu Ala Ala Phe Asn Lys Gly Glu Thr
210 215 220
Ala Met Thr Ile Asn Gly Pro Trp Ala Trp Ser Asn Ile Asp Thr Ser
225 230 235 240
Lys Val Asn Tyr Gly Val Thr Val Leu Pro Thr Phe Lys Gly Gln Pro
245 250 255
Ser Lys Pro Phe Val Gly Val Leu Ser Ala Gly Ile Asn Ala Ala Ser
260 265 270
Pro Asn Lys Glu Leu Ala Lys Glu Phe Leu Glu Asn Tyr Leu Leu Thr
275 280 285
Asp Glu Gly Leu Glu Ala Val Asn Lys Asp Lys Pro Leu Gly Ala Val
290 295 300
Ala Leu Lys Ser Tyr Glu Glu Glu Leu Ala Lys Asp Pro Arg Ile Ala
305 310 315 320
Ala Thr Met Glu Asn Ala Gln Lys Gly Glu Ile Met Pro Asn Ile Pro
325 330 335
Gln Met Ser Ala Phe Trp Tyr Ala Val Arg Thr Ala Val Ile Asn Ala
340 345 350
Ala Ser Gly Arg Gln Thr Val Asp Glu Ala Leu Lys Asp Ala Gln Thr
355 360 365
Asn Ser Gly Ser Gly Gly Ser Gly Gly Glu Ser Leu Phe Lys Gly Pro
370 375 380
Arg Asp Tyr Asn Pro Ile Ser Ser Ser Ile Cys His Leu Thr Asn Glu
385 390 395 400
Ser Asp Gly His Thr Thr Ser Leu Tyr Gly Ile Gly Phe Gly Pro Phe
405 410 415
Ile Ile Thr Asn Lys His Leu Phe Arg Arg Asn Asn Gly Thr Leu Val
420 425 430
Val Gln Ser Leu His Gly Val Phe Lys Val Lys Asp Thr Thr Thr Leu
435 440 445
Gln Gln His Leu Val Asp Gly Arg Asp Met Ile Ile Ile Arg Met Pro
450 455 460
Lys Asp Phe Pro Pro Phe Pro Gln Lys Leu Lys Phe Arg Glu Pro Gln
465 470 475 480
Arg Glu Glu Arg Ile Cys Leu Val Thr Thr Asn Phe Gln Thr Lys Ser
485 490 495
Met Ser Ser Met Val Ser Asp Thr Ser Cys Thr Phe Pro Ser Gly Asp
500 505 510
Gly Ile Phe Trp Lys His Trp Ile Gln Thr Lys Asp Gly Gln Cys Gly
515 520 525
Ser Pro Leu Val Ser Thr Arg Asp Gly Phe Ile Val Gly Ile His Ser
530 535 540
Ala Ser Asn Phe Thr Asn Thr Asn Asn Tyr Phe Thr Ser Val Pro Lys
545 550 555 560
Asn Phe Met Glu Leu Leu Thr Asn Gln Glu Ala Gln Gln Trp Val Ser
565 570 575
Gly Trp Arg Leu Asn Ala Asp Ser Val Leu Trp Gly Gly His Lys Val
580 585 590
Phe Met Val Lys Pro Glu Glu Pro Phe Gln Pro Val Lys Glu Ala Thr
595 600 605
Gln Leu Met Asn Arg Arg Arg Arg Arg
610 615

Claims (10)

1.一种重组烟草蚀纹病毒TEV酶，其特征在于，所述重组TEV酶由TEV蛋白酶突变序列、N端融合的CL7标签和C端融合的多聚精氨酸标签构成，所述TEV蛋白酶突变序列中的突变为以下三种情形之一：
（1）S219V，即TEV蛋白酶的第219位氨基酸残基由丝氨酸突变为缬氨酸；
（2）S219V和S153N，即TEV蛋白酶的第219位氨基酸残基由丝氨酸突变为缬氨酸和TEV蛋白酶的第153位氨基酸残基由丝氨酸突变为天冬酰胺；
（3）T17S、N68D、I77V、S219N、L56V和S135G，即TEV蛋白酶的第17位氨基酸残基由苏氨酸突变为丝氨酸、TEV蛋白酶的第68位氨基酸残基由天冬酰胺突变为天冬氨酸、TEV蛋白酶的第77位氨基酸残基由异亮氨酸突变为缬氨酸、TEV蛋白酶的第219位氨基酸残基由丝氨酸突变为天冬酰胺、TEV蛋白酶的第56位氨基酸残基由亮氨酸突变为缬氨酸和TEV蛋白酶的第135位氨基酸残基由丝氨酸突变为甘氨酸。
2.根据权利要求1所述的一种重组烟草蚀纹病毒TEV酶，其特征在于，所述CL7标签为依次顺式连接的His8-CL7-GGS多肽或His6-CL7-GGS多肽。
3.根据权利要求1所述的一种重组烟草蚀纹病毒TEV酶，其特征在于，所述多聚精氨酸标签的多肽序列为RRRGRRRGRRRG。
4.根据权利要求1所述的一种重组烟草蚀纹病毒TEV酶，其特征在于，所述重组TEV酶的氨基酸序列如SEQ ID NO.2所示。
5.一种多核苷酸，其特征在于，所述多核苷酸编码如权利要求1-4任一所述的重组烟草蚀纹病毒TEV酶。
6.根据权利要求5所述的一种多核苷酸，其特征在于，所述多核苷酸的序列如SEQ IDNO.3所示。
7.一种重组质粒，其特征在于，所述重组质粒为含有如权利要求5-6任一所述的多核苷酸且能够翻译表达出如权利要求1-4任一所述重组烟草蚀纹病毒TEV酶的表达载体。
8.根据权利要求7所述的一种重组质粒，其特征在于，所述表达载体为pET-28a载体。
9.一种重组工程菌，其特征在于，所述重组工程菌包含如权利要求7所述重组质粒。
10.根据权利要求9所述的重组工程菌，其特征在于，所述工程菌为大肠杆菌BL21(DE3)或T7 Express。
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