CN110714020B - 一种高效简便纯化蛋白的方法 - Google Patents

Abstract

传统的柱层析纯化重组蛋白的工艺生产成本较高，步骤繁琐。基于亲和标签的非色谱纯化方法可降低成本，提高靶蛋白产量，但标签去除成本较高。为解决上述问题，本发明提出了一种仅需离心和裂解步骤的简便、高效的纯化方法。该方法使用了CipA包涵体蛋白和内含肽蛋白(Synechocystis sp.PCC6803 DnaB，Ssp DnaB)，利用CipA的自组装功能和DnaB的在弱酸条件下的自裂解功能来实现蛋白纯化。其中靶蛋白可以是要表达和纯化的任意一种蛋白，且纯化后纯度可达95％以上。此外，本发明还对CipA和DnaB之间的连接肽进行了优化，进一步提高了DnaB的裂解效率，同时降低了CipA对靶蛋白活性的影响。该方法有潜力应用于酶的工业化生产。关键词：蛋白质纯化方法，包涵体蛋白CipA，自断裂蛋白DnaB。

Description

一种高效简便纯化蛋白的方法

技术领域

本发明涉及一种表达包涵体蛋白CipA，一种自裂解的内含肽和目标蛋白eGFP的质粒pET-28a-cipA-dnaB-eGFP。本发明还涉及该质粒的构建方法及目标蛋白的纯化方法，属于生物工程技术领域。

背景技术

随着分子生物学、细胞工程和发酵工程的发展，重组蛋白的表达效率显著提高。尽管取得了这样的成功，开发一种大规模、简便且高效的蛋白纯化方法仍然是一项巨大的挑战。在酶催化过程中，酶纯度是降低生产所需产物成本的一个必要环节。许多研究提到使用不同的方法纯化靶蛋白，例如亲和纯化^[1,2]，使用自聚集纯化标签^[3-5]和自我裂解标签(内含肽)^[6,7]等。在这些方法中，亲和纯化在纯化过程中占主导地位，并以其操作简单和纯化效率高仍作为蛋白质纯化的首选。常用的亲和标签包括MBP，His和GST^[8,9]。但亲和纯化具有许多缺点。例如，亲和标签会降低目标酶的催化效率^[9]。另外，亲和标签的切除操作复杂且造价高。尽管相应的亲和树脂可以通过公司购买,并且能达到很高的纯度，但在利用这些方法处理大量的样品时,不得不考虑其高昂的树脂成本和缓慢的纯化速度。

多年来，生物聚合物已被应用于开发低成本且高效的非色谱纯化方法，经实践证实其具有良好的选择性和较高的产率。在大多数情况下，利用生物聚合物作为纯化标签，可以诱导被标记的融合蛋白在特定的化学或物理条件下形成具有高度选择性的聚集体。报道的标签包括弹性蛋白样多肽(ELP)、毒素重复(RTX)结构域和ELK16^[10-13]。其中尤其是ELP标签，它可诱导蛋白聚集。与其他纯化标签一样，这些标签在纯化过程中有时需要被除去，对于可能影响靶蛋白性质的大标签来说，这一步骤尤其重要。常用的标签去除方法包括使用蛋白酶如Xa因子、TEV蛋白酶和鼻病毒3C蛋白酶^[14–16]，但蛋白酶需要额外的纯化步骤来去除，这些纯化步骤会对靶蛋白造成一定的损害，如解离反应可能导致靶蛋白的非特异性裂解或在靶蛋白上留下少量的“残基”氨基酸。使用大量酶去除蛋白标记所带来的高成本严重地限制了它们的应用。据报道，具有自我剪切功能的内含肽已被用于标签去除。内含肽可通过编码基因插入到标记的靶蛋白序列中，其位点特异性自我切割的功能可实现将标签从靶蛋白质上去除的效果。因此，与蛋白酶相比，内含肽在时间成本和生产成本方面具有更高的效益。

为了简化蛋白质纯化操作步骤并降低亲和标签对靶蛋白的影响，本发明提出了一种由CipA蛋白和内含肽介导的纯化方法。CipA是一种含有104个氨基酸的小蛋白质，存在于发光杆菌Photorhabdus luminescens(P.luminescens)中，可以在菌体中自发组装成包涵体(PCIs)。此外，将目标蛋白融合在CipA的C端，融合的蛋白仍可保持其活性。本发明将CipA与靶蛋白P融合，为了从重组蛋白中除去CipA并获得没有任何标签的靶蛋白，在CipA和靶蛋白P之间插入了小内含肽(Synechocystis sp.PCC6803DnaB,Ssp DnaB)以产生CipA-DnaB-PPCIs。DnaB可以在低pH条件下通过其C端裂解与融合的靶蛋白分离。此外，为了提高DnaB自裂解的效率并在上清液中获得更多的靶蛋白，本发明在CipA和DnaB之间插入不同的连接肽。本发明提出了一种新颖简单的蛋白质纯化方法，可以降低所需蛋白质的工业化生产成本。

本发明利用此方法纯化了六种目标蛋白，包括增强型绿色荧光蛋白(eGFP)、β-半乳糖苷酶(β-Gal)、乙酰乳酸合酶(AlsS)、乙酮醇酸还原异构酶(IlvC)、二羟酸脱水酶(IlvD)及丙酮酸脱羧酶(Kivd)，并成功利用纯化的酶在体外实现了异丁醇的生产。

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发明内容

本发明的目的在于提供一种简单高效的纯化靶蛋白的方法，该方法中使用到质粒pET-28a-cipA-dnaB-eGFP，其表达产物CipA-DnaB-eGFP PCIs，改变pH后，可通过内含肽DnaB的自裂解将靶蛋白与包涵体蛋白分离，从而达到纯化的效果。

本发明的另一个目的是提供上述质粒的构建方法和蛋白纯化方法，其包括下述步骤：

(1)构建质粒pET-28a-cipA-dnaB-eGFP，采用OE-PCR法合成了基因cipA(NCBI：CAE138691)、dnaB(PDB：1MI8_A)和eGFP(NCBI：AHK23750.1)，通过OE-PCR分别将cipA和eGFP连接到dnaB的5’端和3’端。通过Gibson组装将cipA-dnaB-eGFP克隆到pET-28a载体上。随后，将PCR产物转化至大肠杆菌XL10-Gold，并通过DNA测序验证质粒。

(2)表达蛋白CipA-DnaB-eGFP，将质粒pET-28a-cipA-dnaB-eGFP导入大肠杆菌BL21(DE3)中，挑取单菌落接种于5mL LB培养基中，加入50μg/mL卡那霉素，37℃过夜培养获得种子液。次日，将种子液接种于200mL新鲜TB培养基中，37℃培养2h左右，使OD₆₀₀达到0.5-0.8，在培养液中加入0.5mM异丙基-β-D-硫半乳糖苷(IPTG)，30℃培养10h。为防止代谢产物使培养基pH下降，在0h、2h、4h和6h分别向培养液中加入25mM HEPES(pH＝8.5)。

(3)获得重组蛋白，将(2)中表达完蛋白的发酵液，离心(4000×g，4℃，20min)，弃上清，获得菌体，加入50mL缓冲液(50mM Tris-HCl，pH 7.5)重悬，超声破碎后离心(10000×g，4℃，20min)，弃上清，最终获得CipA-DnaB-eGFP PCIs。

(4)通过DnaB裂解纯化eGFP，用50mL缓冲液(50mM Tris-HCl，pH＝6.5)洗涤CipA-DnaB-eGFP PCIs两次后重悬，23℃放置18h，使内含肽自裂解，将裂解混合物离心(10000×g，4℃，20min)。其中CipA-DnaB为沉淀，eGFP为上清液中的纯蛋白，整个纯化过程如图1所示。通过SDS-PAGE验证eGFP的含量和纯度。SDS-PAGE浓缩胶和分离胶配方为：5％浓缩胶(3.4mL ddH₂O，830μL 30％丙烯酰胺，630μL 1M Tris-HCl(pH 6.8)，50μL 10％SDS，50μL10％过硫酸铵，5μL TEMED)和10％分离胶(4mL ddH₂O，3.3mL 30％丙烯酰胺，2.5mL 1.5MTris-HCl)，100μL 10％SDS，100μL 10％过硫酸铵，5μL TEMED。

表1：本发明所用到的菌株及质粒

该纯化方法的优化：为了提高DnaB断裂的效率并在上清液中获得更多单一的靶蛋白P，本方法在基因cipA和dnaB序列之间插入了不同的连接肽。使用pET-28a-cipA-dnaB-eGFP作为模板，并在靶蛋白基因p的5’端使用具有“GRA”三个氨基酸编码序列的引物。随后，将PCR产物转化至大肠杆菌XL10-Gold中使其自连接，得到重组质粒pET-28a-CIG1，随后通过测序验证重组质粒。另外，质粒pET-28a-cipA-EPPPL-dnaB-GRA-eGFP(pET-28a-CIG2)，pET-28a-cipA-EPPPLPPPLPPPL-dnaB-GRA-eGFP(pET-28a-CIG3)，pET-28a-cipA-GSGSGS-dnaB-GRA-eGFP(pET-28a-CIG4)，pET28a-cipA-GGGGSGGGGSGGGGS-dnaB-GRA-eGFP(pET-28a-CIG5)，与pET-28a-CIG1构建过程相似。实验验证表达质粒pET28a-cipA-GGGGSGGGGSGGGGS-dnaB-GRA-eGFP(pET-28a-CIG5)所得产物的裂解效果最好，得到的纯化的靶蛋白eGFP最多。

附图说明：

图1使用该方法纯化蛋白的原理流程图，其中靶蛋白为eGFP。

图2使用该方法纯化蛋白的操作流程图，其中靶蛋白为eGFP。

图3大肠杆菌BL21(DE3)(pET-28a-cipA-dnaB-eGFP)表达产物CipA-DnaB-eGFP及CipA-DnaB和eGFP的SDS-PAGE电泳图。泳道1：180kDa蛋白Marker；泳道2：超声破碎后的全细胞蛋白；泳道3：破碎混合物离心所得沉淀；泳道4：破碎混合物离心所得上清；泳道5：内含肽裂解后离心所得沉淀；泳道6：内含肽裂解后离心所得上清。

图4大肠杆菌BL21(DE3)(pET-28a-cipA-dnaB-adhP)表达产物CipA-DnaB-AdhP及CipA-DnaB和AdhP的SDS-PAGE电泳图。泳道1：180kDa蛋白Marker；泳道2：超声破碎后的全细胞蛋白；泳道3：破碎混合物离心所得沉淀；泳道4：破碎混合物离心所得上清；泳道5：内含肽裂解后离心所得沉淀；泳道6：内含肽裂解后离心所得上清。

图5大肠杆菌BL21(DE3)(pET-28a-cipA-dnaB-MBP)表达产物CipA-DnaB-MBP及CipA-DnaB和MBP的SDS-PAGE电泳图。泳道1：180kDa蛋白Marker；泳道2：超声破碎后的全细胞蛋白；泳道3：破碎混合物离心所得沉淀；泳道4：破碎混合物离心所得上清；泳道5：内含肽裂解后离心所得沉淀；泳道6：内含肽裂解后离心所得上清。

序列表：

序列1，包涵体蛋白CipA的氨基酸序列；

序列2，内含肽DnaB的氨基酸序列；

序列3，增强型绿色荧光蛋白eGFP的氨基酸序列；

序列4，β-半乳糖苷酶(β-Gal)的氨基酸序列；

序列5，纤维素结合蛋白(MBP)的氨基酸序列；

序列6，乙酰乳酸合酶(AlsS)的氨基酸序列；

序列7，乙酮醇酸还原异构酶(IlvC)的氨基酸序列；

序列8，二羟酸脱水酶(IlvD)的氨基酸序列；

序列9，丙酮酸脱羧酶(Kivd)的氨基酸序列；

序列10，丙酮酸脱羧酶(AdhP)的氨基酸序列。

具体实施方式

以下实例是对本发明的进一步说明，并不构成对本发明实质内容的限制。

实施例1

增强型绿色荧光蛋白eGFP的纯化

(1)构建质粒pET-28a-cipA-dnaB-eGFP，采用OE-PCR法合成了基因cipA(NCBI：CAE138691)、dnaB(PDB：1MI8_A)和eGFP(NCBI：AHK23750.1)，通过OE-PCR分别将cipA和eGFP连接到dnaB的5’端和3’端。通过Gibson组装将cipA-dnaB-eGFP克隆到pET-28a载体上。随后，将PCR产物转化至大肠杆菌XL10-Gold，并通过DNA测序验证质粒。

(2)表达蛋白CipA-DnaB-eGFP，将质粒pET-28a-cipA-dnaB-eGFP导入大肠杆菌BL21(DE3)中，挑取单菌落接种于5mL LB培养基中，加入50μg/mL卡那霉素，37℃过夜培养获得种子液。次日，将种子液接种于200mL新鲜TB培养基中，37℃培养2h左右，使OD₆₀₀达到0.5-0.8，在培养液中加入0.5mM异丙基-β-D-硫半乳糖苷(IPTG)，30℃培养10h。为防止代谢产物使培养基pH下降，在0h、2h、4h和6h分别向培养液中加入25mM HEPES(pH＝8.5)。

(3)获得重组蛋白，将(2)中表达完蛋白的发酵液，离心(4000×g，4℃，20min)，弃上清，获得菌体，加入50mL缓冲液(50mM Tris-HCl，pH 7.5)重悬，超声破碎后离心(10000×g，4℃，20min)，弃上清，最终获得CipA-DnaB-eGFP PCIs。

(4)通过DnaB裂解纯化eGFP，用50mL缓冲液(50mM Tris-HCl，pH＝6.5)洗涤CipA-DnaB-eGFP PCIs两次后重悬，23℃放置18h，使内含肽自裂解，将裂解混合物离心(10000×g，4℃，20min)。其中CipA-DnaB为沉淀，eGFP为上清液中的纯蛋白，整个纯化过程如图1所示。通过SDS-PAGE验证eGFP的含量和纯度。SDS-PAGE浓缩胶和分离胶配方为：5％浓缩胶(3.4mL ddH₂O，830μL 30％丙烯酰胺，630μL 1M Tris-HCl(pH 6.8)，50μL 10％SDS，50μL10％过硫酸铵，5μL TEMED)和10％分离胶(4mL ddH₂O，3.3mL 30％丙烯酰胺，2.5mL 1.5MTris-HCl)，100μL 10％SDS，100μL 10％过硫酸铵，5μL TEMED。

蛋白表达的培养基组成为：酵母提取物24g/L，蛋白胨12g/L，KH₂PO₄12.54g/L，K₂HPO₄ 2.31g/L，甘油4mL/L，高温高压灭菌。

裂解缓冲液组成为：50mM Tris，10mM DTT，HCl调至pH＝6.5

实施例2

β-半乳糖苷酶(β-Gal)的纯化

(1)构建质粒pET-28α-cipA-dnaB-lacZ(pET-28α-CIZ)，采用OE-PCR法合成了基因cipA(NCBI：CAE138691)、dnaB(PDB：1MI8_A)和lacZ(NCBI：945006)。然后，通过OE-PCR分别将cipA和lacZ连接到dnaB的5’端和3’端。然后通过Gibson组装将cipA-dnaB-lacZ片段克隆到pET-28a载体上。随后，将PCR产物转化大肠杆菌XL10-Gold，测序验证重组质粒。

(2)表达融合蛋白CipA-DnaB-β-Gal，将pET-28α-CIZ转化至大肠杆菌BL21(DE3)中，并将单个菌落接种于5mL LB培养基中，加入50μg/mL卡那霉素，在37°С培养过夜后将培养液接种于200mL新鲜TB培养基中。37℃培养2h后使OD₆₀₀达到0.5-0.8，加入0.5mM异丙基-β-D-硫半乳糖苷(IPTG)，30℃培养10h。为了防止培养基中的介质酸化和培养液pH下降，在培养0h、2h、4h和6h时加入25mM HEPES(pH 8.5)。

(3)获得重组蛋白，将(2)中表达完蛋白的发酵液，离心(4000×g，4℃，20min)，弃上清，获得菌体，加入50mL缓冲液(50mM Tris-HCl，pH 7.5)重悬，超声破碎后离心(10000×g，4℃，20min)，弃上清，最终获得CipA-DnaB-β-Gal PCIs。

(4)纯化β-Gal，用50mM裂解缓冲液(50mM Tris-HCl，pH 6.5)洗涤沉淀CipA-DnaB-β-Gal PCIs两次，最后用裂解缓冲液重悬，使其浓度达到OD₆₀₀为1，然后在23℃下放置18h，DnaB的C端裂解。将裂解混合物离心(10000×g，4℃，20min)。CipA-DnaB保留在沉淀中，β-Gal则作为上清液中的唯一蛋白被纯化出来。通过SDS-PAGE电泳确定β-Gal的含量和纯度。

酶活性的测定，参照Gil-Martín等人的方法略加改进。在总体积为1mL的柠檬酸反应缓冲液(0.1M柠檬酸用NaOH调至pH 4.5)，体系中含0.8μmol的底物ONPG和40μmolβ-巯基乙醇(临用前配制)，适量纯化后的酶液。在37℃孵育30min后，用0.5M冷碳酸钠1mL终止反应，用紫外分光光度计于405nm波长测定吸光度值，同时设定底物对照和酶液对照。产物邻硝基苯酚(ONP)显黄色，在405nm处有最大吸收峰。

酶活性计算公式：U(β-Gal)＝A/(0.777×30)×m。

式中：A为反应生成的ONP吸光度值，A＝A(反应液)-A(底物)-A(酶提取液)；0.777为1μmol邻硝基苯酚(ONP)的吸光度值；m为反应体系中的酶蛋白量，单位是mg。酶活力单位U定义为标准条件下(pH 4.5，37℃)每min催化生成1μmol产物邻硝基苯酚(ONP)所需的酶量为1个单位。

蛋白表达的培养基组成为：酵母提取物24g/L，蛋白胨12g/L，KH₂PO₄12.54g/L，K₂HPO₄ 2.31g/L，甘油4ml/L，蒸馏水定容至1L，高温高压灭菌。

裂解缓冲液组成为：50mM Tris，10mM DTT，HCl调至pH＝6.5

实施例3

麦芽糖结合蛋白(MBP)的纯化

(1)构建质粒pET-28α-cipA-dnaB-MBP(pET-28α-CIM)，采用OE-PCR法合成了基因cipA(NCBI：CAE138691)、dnaB(PDB：1MI8_A)和MBP(Gene ID:4155)。然后，通过OE-PCR分别将cipA和MBP连接到dnaB的5’端和3’端。然后通过Gibson组装将cipA-dnaB-MBP片段克隆到pET-28a载体上。随后，将PCR产物转化大肠杆菌XL10-Gold，测序验证重组质粒。

(2)表达融合蛋白CipA-DnaB-MBP，将pET-28α-CIM转化至大肠杆菌BL21(DE3)中，并将单个菌落接种于5mL LB培养基中，加入50μg/mL卡那霉素，在37℃培养过夜后将培养液接种于200mL新鲜TB培养基中。37℃培养2h后使OD₆₀₀达到0.5-0.8，加入0.5mM异丙基-β-D-硫半乳糖苷(IPTG)，30℃培养10h。为了防止培养基中的介质酸化和培养液pH下降，在培养0h、2h、4h和6h时加入25mM HEPES(pH 8.5)。

(3)获得重组蛋白，将(2)中表达完蛋白的发酵液，离心(4000×g，4℃，20min)，弃上清，获得菌体，加入50mL缓冲液(50mM Tris-HCl，pH 7.5)重悬，超声破碎后离心(10000×g，4℃，20min)，弃上清，最终获得CipA-DnaB-MBP PCIs。

(4)纯化MBP，用50mM裂解缓冲液(50mM Tris-HCl，pH 6.5)洗涤沉淀CipA-DnaB-MBP PCIs两次，最后用裂解缓冲液重悬，使其浓度达到OD₆₀₀为1，然后在23℃下放置18h，DnaB的C端裂解。将裂解混合物离心(10000×g，4℃，20min)。CipA-DnaB保留在沉淀中，MBP则作为上清液中的唯一蛋白被纯化出来。通过SDS-PAGE电泳确定MBP的含量和纯度。

蛋白表达的培养基组成为：酵母提取物24g/L，蛋白胨12g/L，KH₂PO₄12.54g/L，K₂HPO₄ 2.31g/L，甘油4ml/L，蒸馏水定容至1L，高温高压灭菌。

裂解缓冲液组成为：50mM Tris，10mM DTT，HCl调至pH＝6.5

实施例4

乙醇脱氢酶(AdhP)的纯化

(1)构建质粒pET-28α-cipA-dnaB-adhP(pET-28α-CIP)，采用OE-PCR法合成了基因cipA(NCBI：CAE138691)、dnaB(PDB：1MI8_A)和adhP(Gene ID:946036)。然后，通过OE-PCR分别将cipA和adhP连接到dnaB的5’端和3’端。然后通过Gibson组装将cipA-dnaB-adhP片段克隆到pET-28a载体上。随后，将PCR产物转化大肠杆菌XL10-Gold，测序验证重组质粒。

(2)表达融合蛋白CipA-DnaB-AdhP，将pET-28α-CIP转化至大肠杆菌BL21(DE3)中，并将单个菌落接种于5mL LB培养基中，加入50μg/mL卡那霉素，在37℃培养过夜后将培养液接种于200mL新鲜TB培养基中。37℃培养2h后使OD₆₀₀达到0.5-0.8，加入0.5mM异丙基-β-D-硫半乳糖苷(IPTG)，30℃培养10h。为了防止培养基中的介质酸化和培养液pH下降，在培养0h、2h、4h和6h时加入25mM HEPES(pH 8.5)。

(3)获得重组蛋白，将(2)中表达完蛋白的发酵液，离心(4000×g，4℃，20min)，弃上清，获得菌体，加入50mL缓冲液(50mM Tris-HCl，pH 7.5)重悬，超声破碎后离心(10000×g，4℃，20min)，弃上清，最终获得CipA-DnaB-AdhP PCIs。

(4)纯化AdhP，用50mM裂解缓冲液(50mM Tris-HCl，pH 6.5)洗涤沉淀CipA-DnaB-AdhP PCIs两次，最后用裂解缓冲液重悬，使其浓度达到OD₆₀₀为1，然后在23℃下放置18h，DnaB的C端裂解。将裂解混合物离心(10000×g，4℃，20min)。CipA-DnaB保留在沉淀中，AdhP则作为上清液中的唯一蛋白被纯化出来。通过SDS-PAGE电泳确定AdhP的含量和纯度。

蛋白表达的培养基组成为：酵母提取物24g/L，蛋白胨12g/L，KH₂PO₄12.54g/L，K₂HPO₄ 2.31g/L，甘油4ml/L，蒸馏水定容至1L，高温高压灭菌。

裂解缓冲液组成为：50mM Tris，10mM DTT，HCl调至pH＝6.5

实施例5

乙酰乳酸合酶(AlsS)、乙酮醇酸还原异构酶(IlvC)、二羟酸脱水酶(IlvD)及丙酮酸脱羧酶(Kivd)的纯化及体外反应

(1)构建质粒pET-28α-cipA-dnaB-GRA-alsS(pET-28α-CIS)、pET-28α-cipA-dnaB-GRA-ilvC(pET-28α-CIC)、pET-28α-cipA-dnaB-GRA-ilvD(pET-28α-CID)和pET-28α-cipA-dnaB-GRA-kivd(pET-28α-CIK)，采用OE-PCR法合成了基因alsS、ilvC、ilvD和kivD。以pET-28a-cipA-dnaB-eGFP为模板，将包含cipA的pET-28a骨架克隆下来，通过Gibson组装分别将4个基因克隆到pET-28a-cipA的骨架中，将连接产物转化至大肠杆菌XL10-Gold，测序验证。

(2)表达融合蛋白CipA-DnaB-AlsS、CipA-DnaB-IlvC、CipA-DnaB-IlvD和CipA-DnaB-Kivd，将质粒pET-28α-CIS、pET-28α-CIC、pET-28α-CID和pET-28α-CIK分别转化大肠杆菌BL21(DE3)，将单菌落接种于5mL LB培养基中，加入50μg/mL卡那霉素，在37℃培养过夜后将培养液接种于200mL新鲜TB培养基中。37℃培养2h后使OD₆₀₀达到0.5-0.8，加入0.5mM异丙基-β-D-硫半乳糖苷(IPTG)，30℃培养10h。为了防止培养基中的介质酸化和培养液pH下降，在培养0h、2h、4h和6h时加入25mM HEPES(pH 8.5)。

(3)获得重组蛋白，将(2)中表达完蛋白的发酵液，离心(4000×g，4℃，20min)，弃上清，获得菌体，加入50mL缓冲液(50mM Tris-HCl，pH 7.5)重悬，超声破碎后离心(10000×g，4℃，20min)，弃上清，最终获得CipA-DnaB-AlsS、CipA-DnaB-IlvC、CipA-DnaB-IlvD及CipA-DnaB-Kivd PCIs。通过SDS-PAGE电泳确定所得产物的含量和纯度。

(4)纯化AlsS、IlvC、IlvD和Kivd，用50mM裂解缓冲液(50mM Tris-HCl，pH 6.5)分别洗涤沉淀CipA-DnaB-AlsS、CipA-DnaB-IlvC、CipA-DnaB-IlvD及CipA-DnaB-Kivd PCIs两次，最后用裂解缓冲液重悬，使其浓度达到OD₆₀₀为1，然后在23℃下放置18h，DnaB的C端裂解。将裂解混合物离心(10000×g，4℃，20min)。CipA-DnaB保留在沉淀中，AlsS、IlvC、IlvD及Kivd则分别作为上清液中的唯一蛋白被纯化出来。通过SDS-PAGE电泳确定AlsS、IlvC、IlvD和Kivd的含量和纯度。

(5)配制异丁醇生成反应体系。异丁醇生成反应体系(2mL)：2mM丙酮酸，1mM NAD⁺，4种酶包括实施例4中AdhP各50μL，室温反应2h。

异丁醇检测，使用配备火焰离子化检测器的Agilent 6890气相色谱仪。毛细管柱为DB-FFAP(30m×0.32mm×0.25μm，安捷伦科技)，色谱条件为80℃，3min，利用增加梯度(115℃/min)增加到230℃，并在230℃保持1min，氮气作为载气。进样器和检测器分别维持在250和280℃。取反应后上清液1μL进样，其中进样分流比为1：30，正戊醇为内标。

在1.9min时获得异丁醇的气相色谱峰，且产量为1.2mM，产率为60％。

序列表

序列1：CipA

MINDMHPSLIKDKDIVDDVMLRSCKIIAMKVMPDKVMQVMVTVLMHDGVCEEMLLKWNLLDNRGMAIYKVLMEALCAKKDVKISTVGKVGPLGCDYINCVEISM

序列2：DnaB

AISGDSLISLASTGKRVSIKDLLDEKDFEIWAINEQTMKLESAKVSRVFCTGKK LVYILKTRLGRTIKATANHRFLTIDGWKRLDELSLKEHIALPRKLESSSLQLSPEIEKLSQSDIYWDSIVSITETGVEEVFDLTVPGPHNFVANDIIVHN

序列3：eGFP

MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGITLGMDELYK

序列4：β-Gal

MTMITDSLAVVLQRRDWENPGVTQLNRLAAHPPFASWRNSEEARTDRPSQQLRSLNGEWRFAWFPAPEAVPESWLECDLPEADTVVVPSNWQMHGYDAPIYTNVTYPITVNPPFVPTENPTGCYSLTFNVDESWLQEGQTRIIFDGVNSAFHLWCNGRWVGYGQDSRLPSEFDLSAFLRAGENRLAVMVLRWSDGSYLEDQDMWRMSGIFRDVSLLHKPTTQISDFHVATRFNDDFSRAVLEAEVQMCGELRDYLRVTVSLWQGETQVASGTAPFGGEIIDERGGYADRVTLRLNVENPKLWSAEIPNLYRAVVELHTADGTLIEAEACDVGFREVRIENGLLLLNGKPLLIRGVNRHEHHPLHGQVMDEQTMVQDILLMKQNNFNAVRCSHYPNHPLWYTLCDRYGLYVVDEANIETHGMVPMNRLTDDPRWLPAMSERVTRMVQRDRNHPSVIIWSLGNESGHGANHDALYRWIKSVDPSRPVQYEGGGADTTATDIICPMYARVDEDQPFPAVPKWSIKKWLSLPGETRPLILCEYAHAMGNSLGGFAKYWQAFRQYPRLQGGFVWDWVDQSLIKYDENGNPWSAYGGDFGDTPNDRQFCMNGLVFADRTPHPALTE AKHQQQFFQFRLSGQTIEVTSEYLFRHSDNELLHWMVALDGKPLASGEVPLDVAPQGKQLIELPELPQPESAGQLWLTVRVVQPNATAWSEAGHISAWQQWRLAENLSVTLPAASHAIPHLTTSEMDFCIELGNKRWQFNRQSGFLSQMWIGDKKQLLTPLRDQFTRAPLDNDIGVSEATRIDPNAWVERWKAAGHYQAEAALLQCTADTLADAVLITTAHAWQHQGKTLFISRKTYRIDGSGQMAITVDVEVASDTPHPARIGLNCQLAQVAERVNWLGLGPQENYPDRLTAACFDRWDLPLSDMYTPYVFPSENGLRCGTRELNYGPHQWRGDFQFNISRYSQQQLMETSHRHLLHAEEGTWLNIDGFHMGIGGDDSWSPSVSAEFQLSAGRYHYQLVWCQK

序列5：MBP蛋白序列

MKIEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLEEKFPQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNGKLIAYPIAVEALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIAADGGYAFKYENGKYDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGETAMTINGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFLENYLLTDEGLEAVNKDKPLGAVALKSYEEELAKDPRIAATMENAQKGEIMPNIPQMSAFWYAVRTAVINAASGRQTVDEALKDAQT

序列6：AlsS

MLTKATKEQKSLVKNRGAELVVDCLVEQGVTHVFGIPGAKIDAVFDALQDKGPEIIVARHEQNAAFMAQAVGRLTGKPGVVLVTSGPGASNLATGLLTANTEGDPVVALAGNVIRADRLKRTHQSLDNAALFQPITKYSVEVQDVKNIPEAVTNAFRIASAGQAGAAFVSFPQDVVNEVTNTKNVRAVAAPKLGPAADDAISAAIAKIQT AKLPVVLVGMKGGRPEAIKAVRKLLKKVQLPFVETYQAAGTLSRDLEDQYFGRIGLFRNQPGDLLLEQADVVLTIGYDPIEYDPKFWNINGDRTIIHLDEIIADIDHAYQPDLELIGDIPSTINHIEHDAVKVEFAEREQKILSDLKQYMHEGEQVPADWKSDRAHPLEIVKELRNAVDDHVTVTCDIGSHAIWMSRYFRSYEPLTLMISNGMQTLGVALPWAIGASLVKPGEKVVSVSGDGGFLFSAMELETAVRLKAPIVHIVWNDSTYDMVAFQQLKKYNRTSAVDFGNIDIVKYAESFGATGLRVESPDQLADVLRQGMNAEGPVIIDVPVDYSDNINLASDKLPKEFGELMKTKAL

序列7：IlvC

MAIELLYDADADLSLIQGRKVAIVGYGSQGHAHSQNLRDSGVEVVIGLREGSKSAEKAKEAGFEVKTTAEAAAWADVIMLLAPDTSQAEIFTNDIEPNLNAGDALLFGHGLNIHFDLIKPADDIIVGMVAPKGPGHLVRRQFVDGKGVPCLIAVDQDPTGTAQALTLSYAAAIGGARAGVIPTTFEAETVTDLFGEQAVLCGGTEELVKVGFEVLTEAGYEPEMAYFEVLHELKLIVDLMFEGGISNMNYSVSDTAEFGGYLSGPRVIDADTKSRMKDILTDIQDGTFTKRLIANVENGNTELEGLRASYNNHPIEETGAKLRDLMSWVKVDARAETA

序列8：IlvD

MIPLRSKVTTVGRNAAGARALWRATGTKENEFGKPIVAIVNSYTQFVPGHVHLKNVGDIVADAVRKAGGVPKEFNTIAVDDGIAMGHGGMLYSLPSREIIADSVEYMVNAHTADAMVCISNCDKITPGMLNAAMRLNIPVVFVSGGPMEAGKAVVVDGVAHAPTDLITAISASASDAVDDAGLAAVEASACPTCGSCSGMFTANSMNCLTEALGLSLPGNGSTLATHAARRALFEKAGETVVELCRRYYGEEDESVLPR GIATKKAFENAMALDMAMGGSTNTILHILAAAQEGEVDFDLADIDELSKNVPCLSKVAPNSDYHMEDVHRAGGIPALLGELNRGGLLNKDVHSVHSNDLEGWLDDWDIRSGKTTEVATELFHAAPGGIRTTEAFSTENRWDELDTDAAKGCIRDVEHAYTADGGLVVLRGNISPDGAVIKSAGIEEELWNFTGPARVVESQEEAVSVILTKTIQAGEVLVVRYEGPSGGPGMQEMLHPTAFLKGSGLGKKCALITDGRFSGGSSGLSIGHVSPEAAHGGVIGLIENGDIVSIDVHNRKLEVQVSDEELQRRRDAMNASEKPWQPVNRNRVVTKALRAYAKMATSADKGAVRQVD

序列9：Kivd

MYTVGDYLLDRLHELGIEEIFGVPGDYNLQFLDQIISRKDMKWVGNANELNASYMADGYARTKKAAAFLTTFGVGELSAVNGLAGSYAENLPVVEIVGSPTSKVQNEGKFVHHTLADGDFKHFMKMHEPVTAARTLLTAENATVEIDRVLSALLKERKPVYINLPVDVAAAKAEKPSLPLKKENSTSNTSDQEILNKIQESLKNAKKPIVITGHEIISFGLEKTVSQFISKTKLPITTLNFGKSSVDEALPSFLGIYNGKLSEPNLKEFVESADFILMLGVKLTDSSTGAFTHHLNENKMISLNIDEGKIFNESIQNFDFESLISSLLDLSEIEYKGKYIDKKQEDFVPSNALLSQDRLWQAVENLTQSNETIVAEQGTSFFGASSIFLKPKSHFIGQPLWGSIGYTFPAALGSQIADKESRHLLFIGDGSLQLTVQELGLAIREKINPICFIINNDGYTVEREIHGPNQSYNDIPMWNYSKLPESFGATEERVVSKIVRTENEFVSVMKEAQADPNRMYWIELILAKEDAPKVLKKMGKLFAEQNKS

序列10：AdhP蛋白序列

MKAAVVTKDHHVDVTYKTLRSLKHGEALLKMECCGVCHTDLHVKNG DFGDKTGVILGHEGIGVVAEVGPGVTSLKPGDRASVAWFYEGCGHCEYCNSGNETLCRSVKNAGYSVDGGMAEECIVVADYAVKVPDGLDSAAASSITCAGVTTYKAVKLSKIRPGQWIAIYGLGGLGNLALQYAKNVFNAKVIAIDVNDEQLKLATEMGADLAINSHTEDAAKIVQEKTGGAHAAVVTAVAKAAFNSAVDAVRAGGRVVAVGLPPESMSLDIPRLVLDGIEVVGSLVGTRQDLTEAFQFAAEGKVVPKVALRPLADINTIFTEMEEGKIRGRMVID

序列表

<110> 北京理工大学

<120> 一种高效简便纯化蛋白的方法

<141> 2019-12-02

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Leu Glu Ala Glu Val Gln Met Cys Gly Glu Leu Arg Asp Tyr Leu Arg

245 250 255

Val Thr Val Ser Leu Trp Gln Gly Glu Thr Gln Val Ala Ser Gly Thr

260 265 270

Ala Pro Phe Gly Gly Glu Ile Ile Asp Glu Arg Gly Gly Tyr Ala Asp

275 280 285

Arg Val Thr Leu Arg Leu Asn Val Glu Asn Pro Lys Leu Trp Ser Ala

290 295 300

Glu Ile Pro Asn Leu Tyr Arg Ala Val Val Glu Leu His Thr Ala Asp

305 310 315 320

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

325 330 335

Arg Ile Glu Asn Gly Leu Leu Leu Leu Asn Gly Lys Pro Leu Leu Ile

340 345 350

Arg Gly Val Asn Arg His Glu His His Pro Leu His Gly Gln Val Met

355 360 365

Asp Glu Gln Thr Met Val Gln Asp Ile Leu Leu Met Lys Gln Asn Asn

370 375 380

Phe Asn Ala Val Arg Cys Ser His Tyr Pro Asn His Pro Leu Trp Tyr

385 390 395 400

Thr Leu Cys Asp Arg Tyr Gly Leu Tyr Val Val Asp Glu Ala Asn Ile

405 410 415

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

420 425 430

Trp Leu Pro Ala Met Ser Glu Arg Val Thr Arg Met Val Gln Arg Asp

435 440 445

Arg Asn His Pro Ser Val Ile Ile Trp Ser Leu Gly Asn Glu Ser Gly

450 455 460

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

465 470 475 480

Pro Ser Arg Pro Val Gln Tyr Glu Gly Gly Gly Ala Asp Thr Thr Ala

485 490 495

Thr Asp Ile Ile Cys Pro Met Tyr Ala Arg Val Asp Glu Asp Gln Pro

500 505 510

Phe Pro Ala Val Pro Lys Trp Ser Ile Lys Lys Trp Leu Ser Leu Pro

515 520 525

Gly Glu Thr Arg Pro Leu Ile Leu Cys Glu Tyr Ala His Ala Met Gly

530 535 540

Asn Ser Leu Gly Gly Phe Ala Lys Tyr Trp Gln Ala Phe Arg Gln Tyr

545 550 555 560

Pro Arg Leu Gln Gly Gly Phe Val Trp Asp Trp Val Asp Gln Ser Leu

565 570 575

Ile Lys Tyr Asp Glu Asn Gly Asn Pro Trp Ser Ala Tyr Gly Gly Asp

580 585 590

Phe Gly Asp Thr Pro Asn Asp Arg Gln Phe Cys Met Asn Gly Leu Val

595 600 605

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

610 615 620

Gln Gln Phe Phe Gln Phe Arg Leu Ser Gly Gln Thr Ile Glu Val Thr

625 630 635 640

Ser Glu Tyr Leu Phe Arg His Ser Asp Asn Glu Leu Leu His Trp Met

645 650 655

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

660 665 670

Val Ala Pro Gln Gly Lys Gln Leu Ile Glu Leu Pro Glu Leu Pro Gln

675 680 685

Pro Glu Ser Ala Gly Gln Leu Trp Leu Thr Val Arg Val Val Gln Pro

690 695 700

Asn Ala Thr Ala Trp Ser Glu Ala Gly His Ile Ser Ala Trp Gln Gln

705 710 715 720

Trp Arg Leu Ala Glu Asn Leu Ser Val Thr Leu Pro Ala Ala Ser His

725 730 735

Ala Ile Pro His Leu Thr Thr Ser Glu Met Asp Phe Cys Ile Glu Leu

740 745 750

Gly Asn Lys Arg Trp Gln Phe Asn Arg Gln Ser Gly Phe Leu Ser Gln

755 760 765

Met Trp Ile Gly Asp Lys Lys Gln Leu Leu Thr Pro Leu Arg Asp Gln

770 775 780

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

785 790 795 800

Arg Ile Asp Pro Asn Ala Trp Val Glu Arg Trp Lys Ala Ala Gly His

805 810 815

Tyr Gln Ala Glu Ala Ala Leu Leu Gln Cys Thr Ala Asp Thr Leu Ala

820 825 830

Asp Ala Val Leu Ile Thr Thr Ala His Ala Trp Gln His Gln Gly Lys

835 840 845

Thr Leu Phe Ile Ser Arg Lys Thr Tyr Arg Ile Asp Gly Ser Gly Gln

850 855 860

Met Ala Ile Thr Val Asp Val Glu Val Ala Ser Asp Thr Pro His Pro

865 870 875 880

Ala Arg Ile Gly Leu Asn Cys Gln Leu Ala Gln Val Ala Glu Arg Val

885 890 895

Asn Trp Leu Gly Leu Gly Pro Gln Glu Asn Tyr Pro Asp Arg Leu Thr

900 905 910

Ala Ala Cys Phe Asp Arg Trp Asp Leu Pro Leu Ser Asp Met Tyr Thr

915 920 925

Pro Tyr Val Phe Pro Ser Glu Asn Gly Leu Arg Cys Gly Thr Arg Glu

930 935 940

Leu Asn Tyr Gly Pro His Gln Trp Arg Gly Asp Phe Gln Phe Asn Ile

945 950 955 960

Ser Arg Tyr Ser Gln Gln Gln Leu Met Glu Thr Ser His Arg His Leu

965 970 975

Leu His Ala Glu Glu Gly Thr Trp Leu Asn Ile Asp Gly Phe His Met

980 985 990

Gly Ile Gly Gly Asp Asp Ser Trp Ser Pro Ser Val Ser Ala Glu Phe

995 1000 1005

Gln Leu Ser Ala Gly Arg Tyr His Tyr Gln Leu Val Trp Cys Gln Lys

1010 1015 1020

<210> 5

<211> 367

<212> PRT

<213> Escherichia coli

<400> 5

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

His Asp Arg Phe Gly Gly Tyr Ala Gln Ser Gly Leu Leu Ala Glu Ile

65 70 75 80

Thr Pro Asp Lys Ala Phe Gln Asp Lys Leu Tyr Pro Phe Thr Trp Asp

85 90 95

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

100 105 110

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

115 120 125

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

130 135 140

Lys Ser Ala Leu Met Phe Asn Leu Gln Glu Pro Tyr Phe Thr Trp Pro

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

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

245 250 255

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

260 265 270

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

275 280 285

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

290 295 300

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

305 310 315 320

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

325 330 335

Met Ser Ala Phe Trp Tyr Ala Val Arg Thr Ala Val Ile Asn Ala Ala

340 345 350

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

355 360 365

<210> 6

<211> 571

<212> PRT

<213> Bacillus subtilis

<400> 6

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

1 5 10 15

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

20 25 30

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

35 40 45

Gln Asp Lys Gly Pro Glu Ile Ile Val Ala Arg His Glu Gln Asn Ala

50 55 60

Ala Phe Met Ala Gln Ala Val Gly Arg Leu Thr Gly Lys Pro Gly Val

65 70 75 80

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

85 90 95

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

100 105 110

Val Ile Arg Ala Asp Arg Leu Lys Arg Thr His Gln Ser Leu Asp Asn

115 120 125

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

130 135 140

Val Lys Asn Ile Pro Glu Ala Val Thr Asn Ala Phe Arg Ile Ala Ser

145 150 155 160

Ala Gly Gln Ala Gly Ala Ala Phe Val Ser Phe Pro Gln Asp Val Val

165 170 175

Asn Glu Val Thr Asn Thr Lys Asn Val Arg Ala Val Ala Ala Pro Lys

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

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

245 250 255

Glu Asp Gln Tyr Phe Gly Arg Ile Gly Leu Phe Arg Asn Gln Pro Gly

260 265 270

Asp Leu Leu Leu Glu Gln Ala Asp Val Val Leu Thr Ile Gly Tyr Asp

275 280 285

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

290 295 300

Ile Ile His Leu Asp Glu Ile Ile Ala Asp Ile Asp His Ala Tyr Gln

305 310 315 320

Pro Asp Leu Glu Leu Ile Gly Asp Ile Pro Ser Thr Ile Asn His Ile

325 330 335

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

340 345 350

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

355 360 365

Asp Trp Lys Ser Asp Arg Ala His Pro Leu Glu Ile Val Lys Glu Leu

370 375 380

Arg Asn Ala Val Asp Asp His Val Thr Val Thr Cys Asp Ile Gly Ser

385 390 395 400

His Ala Ile Trp Met Ser Arg Tyr Phe Arg Ser Tyr Glu Pro Leu Thr

405 410 415

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

420 425 430

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

435 440 445

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

450 455 460

Val Arg Leu Lys Ala Pro Ile Val His Ile Val Trp Asn Asp Ser Thr

465 470 475 480

Tyr Asp Met Val Ala Phe Gln Gln Leu Lys Lys Tyr Asn Arg Thr Ser

485 490 495

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

500 505 510

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

515 520 525

Leu Arg Gln Gly Met Asn Ala Glu Gly Pro Val Ile Ile Asp Val Pro

530 535 540

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

545 550 555 560

Glu Phe Gly Glu Leu Met Lys Thr Lys Ala Leu

565 570

<210> 7

<211> 338

<212> PRT

<213> Escherichia coli

<400> 7

Met Ala Ile Glu Leu Leu Tyr Asp Ala Asp Ala Asp Leu Ser Leu Ile

1 5 10 15

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

20 25 30

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

35 40 45

Arg Glu Gly Ser Lys Ser Ala Glu Lys Ala Lys Glu Ala Gly Phe Glu

50 55 60

Val Lys Thr Thr Ala Glu Ala Ala Ala Trp Ala Asp Val Ile Met Leu

65 70 75 80

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

85 90 95

Pro Asn Leu Asn Ala Gly Asp Ala Leu Leu Phe Gly His Gly Leu Asn

100 105 110

Ile His Phe Asp Leu Ile Lys Pro Ala Asp Asp Ile Ile Val Gly Met

115 120 125

Val Ala Pro Lys Gly Pro Gly His Leu Val Arg Arg Gln Phe Val Asp

130 135 140

Gly Lys Gly Val Pro Cys Leu Ile Ala Val Asp Gln Asp Pro Thr Gly

145 150 155 160

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

165 170 175

Arg Ala Gly Val Ile Pro Thr Thr Phe Glu Ala Glu Thr Val Thr Asp

180 185 190

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

195 200 205

Lys Val Gly Phe Glu Val Leu Thr Glu Ala Gly Tyr Glu Pro Glu Met

210 215 220

Ala Tyr Phe Glu Val Leu His Glu Leu Lys Leu Ile Val Asp Leu Met

225 230 235 240

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

245 250 255

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

260 265 270

Lys Ser Arg Met Lys Asp Ile Leu Thr Asp Ile Gln Asp Gly Thr Phe

275 280 285

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

290 295 300

Gly Leu Arg Ala Ser Tyr Asn Asn His Pro Ile Glu Glu Thr Gly Ala

305 310 315 320

Lys Leu Arg Asp Leu Met Ser Trp Val Lys Val Asp Ala Arg Ala Glu

325 330 335

Thr Ala

<210> 8

<211> 613

<212> PRT

<213> Escherichia coli

<400> 8

Met Ile Pro Leu Arg Ser Lys Val Thr Thr Val Gly Arg Asn Ala Ala

1 5 10 15

Gly Ala Arg Ala Leu Trp Arg Ala Thr Gly Thr Lys Glu Asn Glu Phe

20 25 30

Gly Lys Pro Ile Val Ala Ile Val Asn Ser Tyr Thr Gln Phe Val Pro

35 40 45

Gly His Val His Leu Lys Asn Val Gly Asp Ile Val Ala Asp Ala Val

50 55 60

Arg Lys Ala Gly Gly Val Pro Lys Glu Phe Asn Thr Ile Ala Val Asp

65 70 75 80

Asp Gly Ile Ala Met Gly His Gly Gly Met Leu Tyr Ser Leu Pro Ser

85 90 95

Arg Glu Ile Ile Ala Asp Ser Val Glu Tyr Met Val Asn Ala His Thr

100 105 110

Ala Asp Ala Met Val Cys Ile Ser Asn Cys Asp Lys Ile Thr Pro Gly

115 120 125

Met Leu Asn Ala Ala Met Arg Leu Asn Ile Pro Val Val Phe Val Ser

130 135 140

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

145 150 155 160

His Ala Pro Thr Asp Leu Ile Thr Ala Ile Ser Ala Ser Ala Ser Asp

165 170 175

Ala Val Asp Asp Ala Gly Leu Ala Ala Val Glu Ala Ser Ala Cys Pro

180 185 190

Thr Cys Gly Ser Cys Ser Gly Met Phe Thr Ala Asn Ser Met Asn Cys

195 200 205

Leu Thr Glu Ala Leu Gly Leu Ser Leu Pro Gly Asn Gly Ser Thr Leu

210 215 220

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

225 230 235 240

Val Val Glu Leu Cys Arg Arg Tyr Tyr Gly Glu Glu Asp Glu Ser Val

245 250 255

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

260 265 270

Leu Asp Met Ala Met Gly Gly Ser Thr Asn Thr Ile Leu His Ile Leu

275 280 285

Ala Ala Ala Gln Glu Gly Glu Val Asp Phe Asp Leu Ala Asp Ile Asp

290 295 300

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

305 310 315 320

Asp Tyr His Met Glu Asp Val His Arg Ala Gly Gly Ile Pro Ala Leu

325 330 335

Leu Gly Glu Leu Asn Arg Gly Gly Leu Leu Asn Lys Asp Val His Ser

340 345 350

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

355 360 365

Ser Gly Lys Thr Thr Glu Val Ala Thr Glu Leu Phe His Ala Ala Pro

370 375 380

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

385 390 395 400

Glu Leu Asp Thr Asp Ala Ala Lys Gly Cys Ile Arg Asp Val Glu His

405 410 415

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

420 425 430

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

435 440 445

Asn Phe Thr Gly Pro Ala Arg Val Val Glu Ser Gln Glu Glu Ala Val

450 455 460

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

465 470 475 480

Arg Tyr Glu Gly Pro Ser Gly Gly Pro Gly Met Gln Glu Met Leu His

485 490 495

Pro Thr Ala Phe Leu Lys Gly Ser Gly Leu Gly Lys Lys Cys Ala Leu

500 505 510

Ile Thr Asp Gly Arg Phe Ser Gly Gly Ser Ser Gly Leu Ser Ile Gly

515 520 525

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

530 535 540

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

545 550 555 560

Gln Val Ser Asp Glu Glu Leu Gln Arg Arg Arg Asp Ala Met Asn Ala

565 570 575

Ser Glu Lys Pro Trp Gln Pro Val Asn Arg Asn Arg Val Val Thr Lys

580 585 590

Ala Leu Arg Ala Tyr Ala Lys Met Ala Thr Ser Ala Asp Lys Gly Ala

595 600 605

Val Arg Gln Val Asp

610

<210> 9

<211> 548

<212> PRT

<213> Lactococcus lactis

<400> 9

Met Tyr Thr Val Gly Asp Tyr Leu Leu Asp Arg Leu His Glu Leu Gly

1 5 10 15

Ile Glu Glu Ile Phe Gly Val Pro Gly Asp Tyr Asn Leu Gln Phe Leu

20 25 30

Asp Gln Ile Ile Ser Arg Lys Asp Met Lys Trp Val Gly Asn Ala Asn

35 40 45

Glu Leu Asn Ala Ser Tyr Met Ala Asp Gly Tyr Ala Arg Thr Lys Lys

50 55 60

Ala Ala Ala Phe Leu Thr Thr Phe Gly Val Gly Glu Leu Ser Ala Val

65 70 75 80

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

85 90 95

Val Gly Ser Pro Thr Ser Lys Val Gln Asn Glu Gly Lys Phe Val His

100 105 110

His Thr Leu Ala Asp Gly Asp Phe Lys His Phe Met Lys Met His Glu

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

Glu Ile Leu Asn Lys Ile Gln Glu Ser Leu Lys Asn Ala Lys Lys Pro

195 200 205

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

210 215 220

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

225 230 235 240

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

245 250 255

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

260 265 270

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

275 280 285

Gly Ala Phe Thr His His Leu Asn Glu Asn Lys Met Ile Ser Leu Asn

290 295 300

Ile Asp Glu Gly Lys Ile Phe Asn Glu Ser Ile Gln Asn Phe Asp Phe

305 310 315 320

Glu Ser Leu Ile Ser Ser Leu Leu Asp Leu Ser Glu Ile Glu Tyr Lys

325 330 335

Gly Lys Tyr Ile Asp Lys Lys Gln Glu Asp Phe Val Pro Ser Asn Ala

340 345 350

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

355 360 365

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

370 375 380

Ser Ser Ile Phe Leu Lys Pro Lys Ser His Phe Ile Gly Gln Pro Leu

385 390 395 400

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

405 410 415

Ala Asp Lys Glu Ser Arg His Leu Leu Phe Ile Gly Asp Gly Ser Leu

420 425 430

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

435 440 445

Pro Ile Cys Phe Ile Ile Asn Asn Asp Gly Tyr Thr Val Glu Arg Glu

450 455 460

Ile His Gly Pro Asn Gln Ser Tyr Asn Asp Ile Pro Met Trp Asn Tyr

465 470 475 480

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

485 490 495

Lys Ile Val Arg Thr Glu Asn Glu Phe Val Ser Val Met Lys Glu Ala

500 505 510

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

515 520 525

Glu Asp Ala Pro Lys Val Leu Lys Lys Met Gly Lys Leu Phe Ala Glu

530 535 540

Gln Asn Lys Ser

545

<210> 10

<211> 333

<212> PRT

<213> Escherichia coli

<400> 10

Met Lys Ala Ala Val Val Thr Lys Asp His His Val Asp Val Thr Tyr

1 5 10 15

Lys Thr Leu Arg Ser Leu Lys His Gly Glu Ala Leu Leu Lys Met Glu

20 25 30

Cys Cys Gly Val Cys His Thr Asp Leu His Val Lys Asn Gly Asp Phe

35 40 45

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

50 55 60

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

65 70 75 80

Ser Val Ala Trp Phe Tyr Glu Gly Cys Gly His Cys Glu Tyr Cys Asn

85 90 95

Ser Gly Asn Glu Thr Leu Cys Arg Ser Val Lys Asn Ala Gly Tyr Ser

100 105 110

Val Asp Gly Gly Met Ala Glu Glu Cys Ile Val Val Ala Asp Tyr Ala

115 120 125

Val Lys Val Pro Asp Gly Leu Asp Ser Ala Ala Ala Ser Ser Ile Thr

130 135 140

Cys Ala Gly Val Thr Thr Tyr Lys Ala Val Lys Leu Ser Lys Ile Arg

145 150 155 160

Pro Gly Gln Trp Ile Ala Ile Tyr Gly Leu Gly Gly Leu Gly Asn Leu

165 170 175

Ala Leu Gln Tyr Ala Lys Asn Val Phe Asn Ala Lys Val Ile Ala Ile

180 185 190

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

195 200 205

Leu Ala Ile Asn Ser His Thr Glu Asp Ala Ala Lys Ile Val Gln Glu

210 215 220

Lys Thr Gly Gly Ala His Ala Ala Val Val Thr Ala Val Ala Lys Ala

225 230 235 240

Ala Phe Asn Ser Ala Val Asp Ala Val Arg Ala Gly Gly Arg Val Val

245 250 255

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

260 265 270

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

275 280 285

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

290 295 300

Lys Val Ala Leu Arg Pro Leu Ala Asp Ile Asn Thr Ile Phe Thr Glu

305 310 315 320

Met Glu Glu Gly Lys Ile Arg Gly Arg Met Val Ile Asp

325 330

Claims (2)

1.一种蛋白纯化标签CipA-GGGGSGGGGSGGGGS-DnaB，其特征在于将自组装蛋白CipA，柔性连接肽GGGGSGGGGSGGGGS和内含肽蛋白DnaB顺序融合，其中CipA的氨基酸序列如序列1所示，DnaB的氨基酸序列如序列2所示。

2.一种高效简便纯化蛋白的方法，其质粒构建及蛋白纯化方法如下：

(1)构建质粒pET-28a-cipA-GGGGSGGGGSGGGGS-dnaB-eGFP，采用OE-PCR法合成了基因cipA、GGGGSGGGGSGGGGS、dnaB和eGFP，其中CipA的氨基酸序列如序列1所示，DnaB的氨基酸序列如序列2所示，通过OE-PCR分别将cipA和eGFP连接到dnaB的5’端和3’端，随后将GGGGSGGGGSGGGGS插入到cipA和dnaB之间，通过Gibson组装将cipA-GGGGSGGGGSGGGGS-dnaB-eGFP克隆到pET-28a载体上，随后，将PCR产物转化至大肠杆菌XL10-Gold，并通过DNA测序验证质粒；

(2)表达蛋白CipA-GGGGSGGGGSGGGGS-DnaB-eGFP，将质粒pET-28a-cipA-GGGGSGGGGSGGGGS-dnaB-eGFP转化到大肠杆菌BL21(DE3)中，挑取单菌落接种于5mL LB培养基中，加入50μg/mL卡那霉素，37℃过夜培养获得种子液，次日，将种子液接种于200mL新鲜TB培养基中，37℃培养2h，使OD₆₀₀达到0.5-0.8，在培养液中加入0.5mM异丙基-B-D-硫半乳糖苷，30℃培养10h，为防止代谢产物使培养基pH下降，在0h、2h、4h和6h分别向培养液中加入25mM，pH8.5，HEPES；

(3)获得重组蛋白，将(2)中表达完蛋白的发酵液，4000×g，4℃，离心20min，弃上清，获得菌体，加入50mL，50mM，pH7.5，Tris-HCl缓冲液重悬，超声破碎后，4000×g，4℃，离心20min，弃上清，最终获得CipA-GGGGSGGGGSGGGGS-DnaB-eGFP包涵体；

(4)通过DnaB裂解纯化eGFP，用50mL，50mM，pH6.5，Tris-HCl缓冲液洗涤CipA-GGGGSGGGGSGGGGS-DnaB-eGFP包涵体两次后重悬，23℃放置18h，使内含肽自裂解，将裂解混合物10000×g，4℃，离心20min，其中CipA-GGGGSGGGGSGGGGS-DnaB为沉淀，eGFP为上清液中的纯蛋白，通过SDS-PAGE验证eGFP的含量和纯度。

Images