CN105838694A - 一种融合标签蛋白 - Google Patents
一种融合标签蛋白 Download PDFInfo
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- CN105838694A CN105838694A CN201610331686.1A CN201610331686A CN105838694A CN 105838694 A CN105838694 A CN 105838694A CN 201610331686 A CN201610331686 A CN 201610331686A CN 105838694 A CN105838694 A CN 105838694A
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- Prior art keywords
- ala
- gly
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- asp
- glu
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- 150000001413 amino acids Chemical class 0.000 claims abstract description 27
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- 108010031186 Glycoside Hydrolases Proteins 0.000 claims abstract description 25
- 102000037865 fusion proteins Human genes 0.000 claims abstract description 24
- 108020001507 fusion proteins Proteins 0.000 claims abstract description 24
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- 125000003275 alpha amino acid group Chemical group 0.000 claims 5
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Abstract
本发明公开了一种融合标签蛋白，本发明的融合标签蛋白bff基于耐有机糖苷酶Fru6构建，为截断至耐有机糖苷酶Fru6N端前n个氨基酸截短体，所述n＜452。本发明还提供了基于本发明的融合标签蛋白构建的融合蛋白表达系统，并选择bff312，bff209，bff217，bff243，bff350作为本发明优选的融合标签蛋白，构建融合蛋白表达系统，验证本发明融合标签蛋白的效果。本发明通过融合标签的连接能有效提高后接蛋白的表达及蛋白折叠，有效提高可溶性蛋白量。
Description
技术领域
本发明属于分子生物学技术领域，具体涉及基于耐有机糖苷酶Fru6的新型的融合标签蛋白，以及基于该融合标签的新型融合蛋白表达系统。
背景技术
大肠杆菌因其背景清晰、生长快速、表达量大，易于操作等特点仍为最常用的宿主，但是它也具有很多局限性，例如：一些异源蛋白在大肠杆菌体内表达时表达量很低，一些蛋白表达后易于降解，或者表达的外源蛋白对宿主细胞具有毒性，导致细菌生长停滞或死亡。或者因未能正确折叠而聚合在一起产生大量包涵体。事实上，大规模生产性状确切的活性蛋白仍是重组蛋白技术中的瓶颈问题。重组蛋白质异源表达中有超过半数以上是以包涵体形式存在的。对包涵体进行体外复性实验使其重新折叠的方法不仅复杂，而且难以保证变复性后蛋白结构与正常生理状态下一致。
融合标签的兴起为重组蛋白的生产带来了福音，融合标签技术在提高蛋白质的可溶性表达中得到广泛应用。
融合标签能增加重组蛋白表达量或在蛋白质折叠过程起作用，增强重组蛋白质的可溶性表达，常被用作标签蛋白与目的蛋白融合在一起表达。此类标签蛋白被称为SETs(Solubility-Enhancing Tags)，这类蛋白主要有谷胱甘肽S-转移酶(GST)，麦芽糖结合蛋白(MBP)，硫氧还蛋白A(TrxA)，转录终止抗终止因子(NusA)，蛋白二硫键折叠异构酶(DsbA)等。但此类标签蛋白究竟如何帮助目的蛋白的表达和折叠，机理尚不明确，且这些SETs不能帮助所有的蛋白质表达和折叠。不同蛋白和融合标签的匹配性不同，对于某种难以表达的蛋白，我们可能需要尝试使用不同的SETs以找到合适的表达条件。
发明内容
本发明目的在于提供一种融合标签蛋白bff以及基于此标签蛋白所构建的融合蛋白表达系统。
本发明所提供的融合签蛋白bff以耐有机糖苷酶Fru6为基础构建，耐有机糖苷酶Fru6分子量约为55kDa，来源于耐有机糖苷酶Fru6产生菌Arthrobacter arilaitensisNJEM01(已公开于专利CN102732456B，保藏编号为CCTCC NO：M2012155)，其氨基酸序列如序列表中SEQ ID No：1所示，其中包括长度为53个氨基酸的信号肽。信号肽具有强分泌性介导该酶分泌表达。本发明所述融合标签bff为截断至耐有机糖苷酶Fru6N端前n个氨基酸截短体。
本发明还涉及bff的编码基因，为耐有机糖苷酶Fru6N端n个氨基酸的编码基因，耐有机糖苷酶Fru6编码基因的核苷酸序列如序列表中SEQ ID No.1所示。
截短体bff在大肠杆菌中表达时可溶表达量很高而且性质稳定，故可作为一种潜在的具有增溶作用的标签蛋白。
本发明根据修正后Wilkinson和Harrison法(http：//www.biotech.ou.edu/)预测不同长度的bff截短体可溶性，预测结果如图1所示，预测时要将信号肽去除后再预测，信号肽去除前长度在452以上个的氨基酸的bff截短体均为不溶，因此上述n<452。
根据预测结果，本发明选择截短体bff209，bff217，bff243，bff312，bff350作为本发明的优选融合标签蛋白，验证本发明的技术效果。所述bff209为n＝209的情形，即为截断至耐有机糖苷酶Fru6N端前209个氨基酸截短体，其氨基酸序列和核苷酸序列如序列表中SEQ ID No：3-4所示。同理，bff217，bff243，bff312，bff350分别指n＝217，n＝243，n＝312，n＝350的情形，其氨基酸序列分别对应序列表中SEQ ID No：5-6、SEQ ID No：7-8、SEQ IDNo：9-10、SEQ ID No：11-12。
为进一步方便目的蛋白的表达和纯化，本发明还提供了基于所述融合标签蛋白构建的融合标签蛋白表达系统。改造表达载体pET-28a，构建含有融合标签的克隆及蛋白表达质粒载体pET-28a-bff，为后续获得天然N端的目的蛋白，在基因后面加入肠激酶酶切位点(DDDDK)，在表达融合蛋白后能够切除融合标签。
本发明中应用四种药用蛋白来测试新型融合表达系统的效果，所用四种药用蛋白为衣原体外膜蛋白抗原决定簇rabOmp、血管内皮生长因子(VEGF)与其相应的受体(VEGFR家族)、社区获得性呼吸窘迫综合征毒素(MPE)蛋白和风疹病毒结构蛋白(Rub)蛋白。
衣原体外膜蛋白抗原决定簇对于研究衣原体主要外膜蛋白的抗原表位对于免疫防御和免疫诊断有重要意义。衣原体外膜蛋白抗原决定簇在大肠杆菌中通常以包涵体的形式表达，要通过复杂的复性和纯化才能获得。衣原体外膜蛋白抗原决定簇的蛋白序列长为216个氨基酸，分子量23.8kDa。
血管内皮生长因子(VEGF)与其相应的受体(VEGFR家族)是调节血管生成的关键蛋白，而VEGF/VEGFR信号通路抑制剂(如VEGF/VEGFR的抗体)对治疗病理性血管增生有较好疗效。本研究中的血管内皮生长因子受体2(VEGFR-2)是VEGFR家族中一员，蛋白序列长为275个氨基酸，分子量30.9kDa。
社区获得性呼吸窘迫综合征毒素是肺炎衣原体的致病因子，参与肺炎等疾病过程，能造成宿主细胞空泡化从而导致细胞死亡。社区获得性呼吸窘迫综合征毒素(MPE)蛋白序列长248个氨基酸，分子量为28.0kDa。
风疹病毒抗原的酶联吸附法是目前用来诊断诊断风疹病毒常用方法之一，但是由于酶联吸附法需要敏感性高、杭原性好的抗原。目前市场上生产的抗原多来源于细胞培养抗原，但是细胞培养成本高，因此重组风疹病毒抗原在原核生物中的表达有重要的现实意义。风疹病毒结构蛋白(Rub)蛋白序列长211个氨基酸，分子量为22.2kDa。
本发明提供的融合标签蛋白bff312应用于在大肠杆菌中难以表达的水蛭素时，使其获得了高效分泌表达，总表达量大幅提高，其中99％的融合蛋白可溶性表达，60％以上融合蛋白分泌至培养基中，其中胞外蛋白经肠激酶切除前后，测得水蛭素生物活性分别为13ATU/mL和21ATU/mL，可见融合截短体bff312后水蛭素3得以正确折叠而具有生物活性；本发明提供的融合标签bff312应用于单独以包涵体形式表达的血管内皮生长因子受体VEGFR-2，实现了高效可溶性表达，可溶性蛋白比例高达93％，可溶性蛋白量是0.86g/L为单独表达VEGFR-2时(0.08g/L)的11倍。
本发明提供的bff209，bff217，bff243，bff350应用于衣原体外膜蛋白抗原决定簇rabOmp，可溶性rabOmp蛋白总量均增加，优选的，融合标签bff209后可溶性蛋白比例达81％，可溶性蛋白量为0.72g/L，比单独表达提高了6倍。相比较而言，融合常见的商用融合标签麦芽糖结合蛋白(MBP)和谷胱甘肽S转移酶(GST)，可溶性融合蛋白比例和可溶性蛋白量分别是25％，0.20g/L和24％，0.12g/L，融合本发明的中融合标签bff209的可溶性表达量比其融合MBP和GST的可溶性表达量提高了3.6倍和6倍。
本发明的融合表达系统在表达数种自身难以可溶表达的蛋白如水蛭素3(HV3)，衣原体外膜蛋白抗原决定簇(rabOmp)，血管内皮生长因子受体2(VEGFR-2)，社区获得性呼吸窘迫综合征毒素(MPE)，风疹病毒结构蛋白(Rub)等抗原时均获得了很高的表达量，获得了很好的表达效果。其中实现水蛭素胞外表达。
本发明提供的融合标签蛋白bff扩展了融合标签库，为融合标签的选择提供了更多的可能。
附图说明
图1是不同长度的bff截短体可溶性预测结果图。
图2是BL21/pET-ff53bff诱导表达的SDS-PAGE图谱。
图3是截短体质粒的构建过程示意图。
图4是基因扩增结果图。
图5是BL21/pET-bff513，BL21/pET-bff430诱导表达的SDS-PAGE图谱。
图6是BL21/pET-bff401，BL21/pET-bff312，BL21/pET-bff200诱导表达的SDS-PAGE图谱。
图7是重叠PCR法扩增目的基因步骤图。
图8是实施例2构建pET-bff312hv示意图。
图9a、图9b、图9c是实施例2电泳结果图；图中箭头方向为bff截短体融合蛋白的位置，图注为重组子所包含基因。
图9d是实施例2中水蛭素3(HV3)，血管内皮生长因子受体2(VEGFR-2)单独表达以及融合截短体bff312融合标签时表达情况，和可溶性蛋白质浓度比例图。
图10是本发明所述新型融合标签蛋白重组载体pET-28a-bff209，pET-28a-bff217，pET-28a-bff243，pET-28a-bff350构建示意图。
图11是目的基因扩增结果图，M为2000bp长的Marker；泳道1-4分别为PCR产物bff350，bff243，bff217，和bff219。
图12是重组子菌落PCR鉴定结果图，M为2000bp长的Marker；泳道1-12为含pET-bff350，pET-bff243，pET-bff217和pET-bff219的三个重组子的菌落。
图13是目的基因扩增结果图，M为2000bp长的Marker；泳道1-3为PCR产物vegfr(NdeI,XhoI)；泳道4-5为PCR产物mpe(NdeI,XhoI)；泳道6-8分别为PCR产物rabomp(NdeI,XhoI)，rabomp(NdeI,BamHI)和rabomp(BamHI,XhoI)；泳道9为PCR产物rub(NdeI,XhoI)。
图14a是rabOmp单独表达及融合蛋白标签bff209，bff217，bff243，bff350表达结果对比图。
图14b是rabOmp单独表达及融合蛋白标签bff209，bff217，bff243，bff350表达蛋白含量可溶性蛋白浓度比例图。
图14c是vegfr单独表达及融合蛋白标签bff209，bff217，bff243，bff350表达结果对比图。
图14d是vegfr单独表达及融合蛋白标签bff209，bff217，bff243，bff350表达蛋白含量可溶性蛋白浓度比例图。
图14e是mpe单独表达及融合蛋白标签bff209，bff217，bff243，bff350表达结果对比图。
图14f是mpe单独表达及融合蛋白标签bff209，bff217，bff243，bff350表达蛋白含量可溶性蛋白浓度比例图。
图14g是rub单独表达及融合蛋白标签bff209，bff217，bff243，bff350表达结果对比图。
图14h是rub单独表达及融合蛋白标签bff209，bff217，bff243，bff350表达蛋白含量可溶性蛋白浓度比例图。
上述图中，M为蛋白Marker；C为发酵液上清，S为细胞裂解液上清，ib为细胞裂解液沉淀。
具体实施方式
下面结合附图，通过实施例具体说明本发明提供的新型融合标签蛋白的构建过程以及新型融合标签表达系统的效果，但不以任何方式限制本发明的范围。
实施例中，如实验具体条件未注明，请参照《分子克隆实验指南》(J.萨姆布鲁克D.W拉塞尔著)所述常规实验，或者仪器、试剂供应商所提供的条件。常规质粒构建所涉及的PCR、酶切、连接等实验，以及蛋白质表达所涉及的转化，细菌培养、诱导等实验为本领域研究人员所熟悉，具体实验细节在本发明中不再做详细描述，亦可参照《分子克隆实验指南》。
实施例1：融合标签bff的偶然发现
果糖苷酶Fru6分子量约为55kDa，其野生酶是一种胞外酶，能够高效合成黄酮类化合物葛根素和芒果苷，并能催化低聚果糖的生成。在生物催化、医药中间体的合成中有着广泛的应用价值。在研究糖苷酶Fru6表达模序时偶然发现，该酶有高度可溶性以及分泌性并且性质稳定。将该酶的开放式阅读框(包括自身信号肽)克隆至大肠杆菌中，可实现高效的胞外分泌表达，信号肽具有强分泌性介导该酶分泌表达，但不影响技术效果。
如图2所示为耐有机糖苷酶Fru6重组子BL21/pET-ff53bff诱导表达的SDS-PAGE图谱，显示出果糖苷酶Fru6的高效折叠能力和胞外分泌能力。
本发明所提供的新型融合签蛋白bff以耐有机糖苷酶Fru6为基础构建，来源于耐有机糖苷酶Fru6产生菌Arthrobacter arilaitensis NJEM01(已公开于专利CN102732456B，保藏编号为CCTCC NO：M2012155)。
本实施例考察了bff及其截短体自身折叠及胞外分泌能力，基于原子经济性原则以及期望获得高效有用的融合标签，随机选取了耐有机糖苷酶Fru6N端截短至513，430，401，312，200位氨基酸的截短体。以耐有机糖苷酶Fru6基因pET-ff53bff为模板，以PF，P200R，P312R，P401R，P513R，P430R为引物扩增，引物序列见表1-1，按照表1-2的PCR反应体系和表1-3的PCR反应条件扩增片段，得到的PCR产物为bff截短体基因片段bff513，bff430，bff401，bff312和bff200，如图3，得到两端分别带有Nco I和Xho I酶切位点的bff截短体基因片段。经纯化后的基因片段bff513，bff430，bff401，bff312，bff200与载体pET-28a用限制性内切酶Xho I和Nco I进行双酶切，37℃酶切8h。将这些片段通过酶切位点插入pET-28a载体中，得到表达载体pET-bff513，pET-bff430，pET-bff401，pET-bff312，pET-bff200。产物使用琼脂糖凝胶电泳验证，结果如图4，分别在约1539bp，1290bp，1203bp，936bp和600bp处有明显条带。
表1-1 引物序列
引物序列如表1-1所示，下划线处为Nco I和Xho I限制性酶切位点。
表1-2 PCR反应体系
表1-3 PCR反应条件
经测序验证正确后，提取质粒转化至E.coli BL21(DE3)感受态细胞，得到重组子BL21/pET-bff513，BL21/pET-bff430，BL21/pET-bff401，BL21/pET-bff312，BL21/pET-bff200。重组子经诱导表达6小时后进行SDS-PAGE，见图2，BL21/pET-bff513，BL21/pET-bff430，BL21/pET-bff401，BL21/pET-bff312，BL21/pET-bff200所表达bff截短体的分子量分别为51.3，42.5，39.4，29.5，16.7kDa。
图5显示，截短至513个和430个氨基酸时，分泌至培养基中的蛋白很少并且出现部分包涵体，图6显示，截短至401个和312个氨基酸时胞外表达量增加，而截短至200个氨基酸时总表达量很少，但仍有分泌。就目前几个截短体而言，截至312个氨基酸(截短体bff312)分泌至培养基中量较多，表达量也较高，能够正确折叠，并所表达的蛋白均已可溶形式表达，甚至实现胞外分泌表达，并且没有不可溶的包涵体存在。
鉴于该蛋白及其截短体的高分泌表达量和高度可溶，推测该蛋白截短体能够实现一些小分子量蛋白的分泌表达和一些通常以包涵体形式表达的药物蛋白可溶性表达。并以高效胞外分泌性的截短体bff312为例，作为融合标签进行后续实验研究。
实施例2：融合标签bff312融合水蛭素(HV3)和血管内皮生长因子受体2(VEGFR-2)质粒构建、表达和活性测定
截短体基因bff312可利用重叠PCR法与HV3/VEGFR-2(下文用hv/vegfr表述)融合。设计重叠PCR引物PF，HVR,HVF,P312HV3R,vegfrR,vegfrF,P312vegfrR。融合bff312和hv：使用引物PF与HVR扩出bff312，使用引物HVF与P312HV3R扩出hv，使用引物PF与P312HV3R扩出bff312hv。融合bff312和vegfr：使用引物PF与vegfrR扩出bff312，使用引物vegfrF与P312vegfrR扩出vegfr，使用引物PF与P312vegfrR扩出bff312vegfr，bff312hv与hv，bff312vegfr与vegfr之间有30个碱基序列重叠，反应步骤如图7所示，图中F1为扩增bff312的上游引物PF，R1为扩增bff312的下游引物HVR/vegfrR，F2为扩增hv/vegfr的上游引物HVF/vegfrF，R2为扩增hv/vegfr的下游引物P312HV3R/P312vegfrR。获得bff312hv/bff312vegfr。
图8是构建pET-bff312hv示意图，pET-bff312vegfr构建方式与pET-bff312hv相同。其中得到的基因片段bff312hv上，bff312和hv之间设有能特异性识别氨基酸序列的蛋白酶肠激酶酶切位点(DDDDK)编码基因(bff312vegfr基因片段在bff312和vegfr之间同样设有DDDDK编码基因)。bff312hv和bff312vegfr与载体pET-28a用限制性内切酶Xho I和NcoI进行双酶切，37℃酶切8h。
表2-1 重叠PCR引物
下划线处为Nco I和Xho I限制性酶切位点。
表2-2 Over lap-PCR反应体系
表2-3 Over-lap PCR扩增条件
酶切产物切胶回收后连接，16℃连接过夜。转化至E.coli BL21(DE3)感受态细胞，得到重组子BL21/pET-bff312hv和BL21/pET-bff312vegfr。
将重组子接种至LBK培养基中(含50g/mL卡那霉素)，37℃，200rpm过夜培养，然后以2％的接种量接种至30mL的LBK培养基中，37℃培养2～3h(OD6000.6～0.9)加IPTG至终浓度为1mM，30℃诱导表达6h。取培养液1mL经过4℃，12000rpm离心10min获得上清为发酵液上清(C)，去除上清后的菌泥使用生理盐水重悬后进行超声破碎5min，离心得到的上清为可溶性组分(S)，离心后沉淀使用生理盐水重悬为不溶性组分(ib)。
经上述处理，电泳结果如图9a、图9b和图9c所示，根据图9a、图9b的SDS-PAGE图谱灰度扫描获得可溶性组分比例如图9d所示。
水蛭素3(HV3)蛋白序列长为69个氨基酸，分子量为7.4kDa；融合bff截短体bff312后分子量为37.2kDa。结果显示，在大肠杆菌中难以表达的水蛭素HV3融合含信号肽的bff截短体bff312获得了高效的分泌表达，蛋白总表达量大幅增加，并且99％的融合蛋白以可溶形式表达，60％以上的融合蛋白分泌在培养基中，不仅获得了高效的可溶性表达(0.8g/L的可溶性蛋白)，而且融合蛋白分泌至培养基，大大简化了蛋白的纯化。
本发明中将上诉融合标签bff312融合于水蛭素N端，通过肠激酶切除标签后获得真实的水蛭素N端，并且融合表达后的大部分蛋白被分泌至胞外不易被水解。其中胞外蛋白经肠激酶切除前后，测得水蛭素生物活性分别为13ATU/mL和21ATU/mL，可见融合截短体bff312后水蛭素3得以正确折叠而具有生物活性。
血管内皮生长因子(VEGF)与其相应的受体(VEGFR家族)是调节血管生成的关键蛋白，而VEGF/VEGFR信号通路抑制剂(如VEGF/VEGFR的抗体)对治疗病理性血管增生有较好疗效。血管内皮生长因子受体2(VEGFR-2)是VEGFR家族中一员，蛋白序列长275个氨基酸，分子量为30.9kDa；融合bff截短bff312后分子量为60.4kDa。单独表达时通常以包涵体形式表达，而融合bff截短体bff312后可溶性蛋白比例高达93％，可溶性蛋白量0.86g/L是单独表达VEGFR-2可溶性蛋白量(0.08g/L)的11倍。
实施例3：新型融合蛋白表达系统的构建以及测试
在本实施例中，四种不同的蛋白被用来测试新型融合蛋白表达系统的效果。
(1)新型融合蛋白表达系统pET-bff350，pET-bff243，pET-bff217和pET-bff209的构建。
图10是本发明所述新型融合标签蛋白重组载体pET-28a-bff209，pET-28a-bff217，pET-28a-bff243，pET-28a-bff350构建示意图。pET-bff350，pET-bff243，pET-bff217和pET-bff209的构建同实施例1中截短体载体构建方法。其中选择的酶切位点为NdeI和Nco I，并且bff350，bff243，bff217和bff209的反向引物上设有编码DDDDK的核苷酸序列，以便以后融合目的蛋白后使用特异性识别DDDDK的肠激酶切除融合标签。以Fru6基因pET-ff53bff为模板为模板，用表3-1中引物PF、P350R、P243R、P217R、P209R分别扩增bff截短体基因片段bff350，bff243，bff 217，bff 209，琼脂糖凝胶电泳验证结果如图11所示，分别在约1065bp，744bp，666bp，和642bp处有明显条带，与各截短体基因长度相符。将基因片段和载体pET-28a经过双酶切后，连接后获得重组质粒pET-bff350，pET-bff243，pET-bff217和pET-bff209。
表3-1 引物序列表
将重组质粒pET-bff350，pET-bff243，pET-bff217和pET-bff209转化至感受态细胞，挑选得到的重组子使用相应的引物进行菌落PCR，PCR产物使用琼脂糖凝胶电泳验证，结果如图12所示，泳道1-12分别为含pET-bff350，pET-bff243，pET-bff217和pET-bff209的三个重组子的菌落PCR验证，与各截短体基因长度相符。菌落PCR验证的重组子经测序后表明目的基因己成功连接于载体。
(2)目的蛋白衣原体外膜蛋白rabOmp，风疹病毒结构蛋白Rub，社区获得性呼吸窘迫综合征毒素Mpe，血管内皮生长因子受体2vegfr-2的可溶性表达。
分别以pET-rabomp，pET-vegfr，pET-mpe，pET-rub为模板，用引物分别扩增基因片段vegfr(Nde I，Xho I)，mpe(Nde I，Xho I)，rub(Nde I，Xho I)，rabomp(Nde I，Xho I)，rabomp(Nde I，BamH I)和rabomp(BamH I，Xho I)，对应引物序列见表3-1，产物使用琼脂糖凝胶电泳验证，结果见图13，图中泳道1-3为PCR产物vegfr(NdeI，XhoI)；泳道4-5为PCR产物mpe(NdeI，XhoI)；泳道6-8分别为PCR产物rabomp(NdeI，XhoI)，rabomp(NdeI，BamHI)和rabomp(BamHI，XhoI)；泳道9为PCR产物rub(NdeI，XhoI)。分别在约825bp，744bp，633bp，648bp，648bp，和648bp处有明显条带，与各目的基因长度相符。
按照上述方法扩增得到的基因片段rabomp(Nde I，Xho I)，vegfr(Nde I，Xho I)，rub(Nde I，Xho I)，mpe(Nde I，Xho I)。将经纯化后的PCR产物及载体pET-bff350，pET-bff243，pET-bff217和pET-bff209用限制性内切酶Xho I和Nde I，37℃酶切8h。
酶切产物切胶回收后连接，16℃连接过夜。转化至E.coli BL21(DE3)感受态细胞，得到重组子BL21/pET-bff209rabomp，BL21/pET-bff217rabomp，BL21/pET-bff243rabomp，BL21/pET-bff350rabomp，BL21/pET-bff209vegfr，BL21/pET-bff217vegfr，BL21/pET-bff243vegfr，BL21/pET-bff350vegfr，BL21/pET-bff209mpe，BL21/pET-bff217mpe，BL21/pET-bff243mpe，BL21/pET-bff350mpe，BL21/pET-bff209rub，BL21/pET-bff217rub，BL21/pET-bff243rub，BL21/pET-bff350rub。
经测序验证正确后，将重组子接种于含50g/mL卡那霉素的LB液体培养基中，37℃，200rpm过夜培养，然后接种至新鲜的含50g/mL卡那霉素的LB培养基中，接种量为2％，37℃培养2～3h(OD6000.6～0.9)，加入异丙基硫代半乳糖苷(IPTG)至终浓度为1mM，30℃诱导表达6h。取培养液1mL经过4℃，12000rpm离心10min获得上清为发酵液上清(C)，去除上清后的菌泥使用生理盐水重悬后进行超声破碎5min，离心得到的上清为可溶性组分(S)，离心后沉淀使用生理盐水重悬为不溶性组分(ib)。
利用SDS-PAGE蛋白凝胶电泳分析各融合蛋白的表达情况，结果如图14a-h所示。融合bff截短体bff209，bff217，bff243，bff350后，融合蛋白成熟肽分子量分别为41.8，42.6，45.5，57.5kDa。为探究最佳长度的bff截短体融合标签，将不同长度的bff截短体标签bff209，bff217，bff243，bff350分别融合rabOmp，vegfr-2，mpe，rub进行表达，其可溶性表达量显著增加，不同截短体融合标签应用于rabOmp，vegfr-2，mpe，rub的总结见表3-2。
表3-2 截短体标签用于蛋白表达总结
来源于耐有机糖苷酶Fru6的截短体标签对一些通常以包涵体形式表达的蛋白有显著的增溶左右，并对一些短肽类蛋白可能具有帮助其分泌至培养基的作用。标签的长度可能会带来效果的差异，对于不同的目的蛋白，融合标签可能会不同。本发明所述的新型融合标签蛋白bff对在大肠杆菌中表达异源蛋白及后续纯化有着重要意义。
序列表
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atg gag aga gcg tgt gtc gcg gtc cga gag att gtc cga ttc cat atc 48
Met Glu Arg Ala Cys Val Ala Val Arg Glu Ile Val Arg Phe His Ile
1 5 10 15
gaa caa agg caa aca atc gtg aac aag caa cgc acc aaa cgc ggc att 96
Glu Gln Arg Gln Thr Ile Val Asn Lys Gln Arg Thr Lys Arg Gly Ile
20 25 30
ctt gcc gca gca ttg agc atc gga gcg ctc gga gca act ttg att tcg 144
Leu Ala Ala Ala Leu Ser Ile Gly Ala Leu Gly Ala Thr Leu Ile Ser
35 40 45
ggg cca gcc atg gct gcc acc gaa cca gtg cct ggc ttc ccc cag cca 192
Gly Pro Ala Met Ala Ala Thr Glu Pro Val Pro Gly Phe Pro Gln Pro
50 55 60
acc gag cac acc cag aag gca tac agc ccc acc gac gat ttc acc tcg 240
Thr Glu His Thr Gln Lys Ala Tyr Ser Pro Thr Asp Asp Phe Thr Ser
65 70 75 80
cgt tgg acc cgt gct gac gca aag cag atc aag gcc atg tcc gac ccg 288
Arg Trp Thr Arg Ala Asp Ala Lys Gln Ile Lys Ala Met Ser Asp Pro
85 90 95
aac gcg gga tcg cgt gaa aac tcc atg ccc aag gag tac acc atg cct 336
Asn Ala Gly Ser Arg Glu Asn Ser Met Pro Lys Glu Tyr Thr Met Pro
100 105 110
acc gtg ccc cag gat ttc cct gac atg agc aac gag gaa gtc tgg gtt 384
Thr Val Pro Gln Asp Phe Pro Asp Met Ser Asn Glu Glu Val Trp Val
115 120 125
tgg gat act tgg cca ctg act gac gag cac gcc aac cag tac agc gtt 432
Trp Asp Thr Trp Pro Leu Thr Asp Glu His Ala Asn Gln Tyr Ser Val
130 135 140
aat ggc cag gag atc atc ttc tcg ctg gtt gcc gat cgc gac ctt ggc 480
Asn Gly Gln Glu Ile Ile Phe Ser Leu Val Ala Asp Arg Asp Leu Gly
145 150 155 160
ttc gat gag cgc cac cag tac gca cgc att gga tac ttc tac cgt cca 528
Phe Asp Glu Arg His Gln Tyr Ala Arg Ile Gly Tyr Phe Tyr Arg Pro
165 170 175
gca ggc gtt cct gcc gat gag cgt cct gaa gat ggt ggc tgg acc tat 576
Ala Gly Val Pro Ala Asp Glu Arg Pro Glu Asp Gly Gly Trp Thr Tyr
180 185 190
ggc ggc cag gtc ttc gat gag ggc gtc acc ggc aag atc ttc gag gac 624
Gly Gly Gln Val Phe Asp Glu Gly Val Thr Gly Lys Ile Phe Glu Asp
195 200 205
cag tcc ttc agc cac cag act cag tgg 651
Gln Ser Phe Ser His Gln Thr Gln Trp
210 215
<210> 6
<211> 217
<212> PRT
<213> Artificial
<220>
<223> Synthetic Construct
<400> 6
Met Glu Arg Ala Cys Val Ala Val Arg Glu Ile Val Arg Phe His Ile
1 5 10 15
Glu Gln Arg Gln Thr Ile Val Asn Lys Gln Arg Thr Lys Arg Gly Ile
20 25 30
Leu Ala Ala Ala Leu Ser Ile Gly Ala Leu Gly Ala Thr Leu Ile Ser
35 40 45
Gly Pro Ala Met Ala Ala Thr Glu Pro Val Pro Gly Phe Pro Gln Pro
50 55 60
Thr Glu His Thr Gln Lys Ala Tyr Ser Pro Thr Asp Asp Phe Thr Ser
65 70 75 80
Arg Trp Thr Arg Ala Asp Ala Lys Gln Ile Lys Ala Met Ser Asp Pro
85 90 95
Asn Ala Gly Ser Arg Glu Asn Ser Met Pro Lys Glu Tyr Thr Met Pro
100 105 110
Thr Val Pro Gln Asp Phe Pro Asp Met Ser Asn Glu Glu Val Trp Val
115 120 125
Trp Asp Thr Trp Pro Leu Thr Asp Glu His Ala Asn Gln Tyr Ser Val
130 135 140
Asn Gly Gln Glu Ile Ile Phe Ser Leu Val Ala Asp Arg Asp Leu Gly
145 150 155 160
Phe Asp Glu Arg His Gln Tyr Ala Arg Ile Gly Tyr Phe Tyr Arg Pro
165 170 175
Ala Gly Val Pro Ala Asp Glu Arg Pro Glu Asp Gly Gly Trp Thr Tyr
180 185 190
Gly Gly Gln Val Phe Asp Glu Gly Val Thr Gly Lys Ile Phe Glu Asp
195 200 205
Gln Ser Phe Ser His Gln Thr Gln Trp
210 215
<210> 7
<211> 729
<212> DNA
<213> Artificial
<220>
<223> bff243
<220>
<221> CDS
<222> (1)..(729)
<400> 7
atg gag aga gcg tgt gtc gcg gtc cga gag att gtc cga ttc cat atc 48
Met Glu Arg Ala Cys Val Ala Val Arg Glu Ile Val Arg Phe His Ile
1 5 10 15
gaa caa agg caa aca atc gtg aac aag caa cgc acc aaa cgc ggc att 96
Glu Gln Arg Gln Thr Ile Val Asn Lys Gln Arg Thr Lys Arg Gly Ile
20 25 30
ctt gcc gca gca ttg agc atc gga gcg ctc gga gca act ttg att tcg 144
Leu Ala Ala Ala Leu Ser Ile Gly Ala Leu Gly Ala Thr Leu Ile Ser
35 40 45
ggg cca gcc atg gct gcc acc gaa cca gtg cct ggc ttc ccc cag cca 192
Gly Pro Ala Met Ala Ala Thr Glu Pro Val Pro Gly Phe Pro Gln Pro
50 55 60
acc gag cac acc cag aag gca tac agc ccc acc gac gat ttc acc tcg 240
Thr Glu His Thr Gln Lys Ala Tyr Ser Pro Thr Asp Asp Phe Thr Ser
65 70 75 80
cgt tgg acc cgt gct gac gca aag cag atc aag gcc atg tcc gac ccg 288
Arg Trp Thr Arg Ala Asp Ala Lys Gln Ile Lys Ala Met Ser Asp Pro
85 90 95
aac gcg gga tcg cgt gaa aac tcc atg ccc aag gag tac acc atg cct 336
Asn Ala Gly Ser Arg Glu Asn Ser Met Pro Lys Glu Tyr Thr Met Pro
100 105 110
acc gtg ccc cag gat ttc cct gac atg agc aac gag gaa gtc tgg gtt 384
Thr Val Pro Gln Asp Phe Pro Asp Met Ser Asn Glu Glu Val Trp Val
115 120 125
tgg gat act tgg cca ctg act gac gag cac gcc aac cag tac agc gtt 432
Trp Asp Thr Trp Pro Leu Thr Asp Glu His Ala Asn Gln Tyr Ser Val
130 135 140
aat ggc cag gag atc atc ttc tcg ctg gtt gcc gat cgc gac ctt ggc 480
Asn Gly Gln Glu Ile Ile Phe Ser Leu Val Ala Asp Arg Asp Leu Gly
145 150 155 160
ttc gat gag cgc cac cag tac gca cgc att gga tac ttc tac cgt cca 528
Phe Asp Glu Arg His Gln Tyr Ala Arg Ile Gly Tyr Phe Tyr Arg Pro
165 170 175
gca ggc gtt cct gcc gat gag cgt cct gaa gat ggt ggc tgg acc tat 576
Ala Gly Val Pro Ala Asp Glu Arg Pro Glu Asp Gly Gly Trp Thr Tyr
180 185 190
ggc ggc cag gtc ttc gat gag ggc gtc acc ggc aag atc ttc gag gac 624
Gly Gly Gln Val Phe Asp Glu Gly Val Thr Gly Lys Ile Phe Glu Asp
195 200 205
cag tcc ttc agc cac cag act cag tgg tcc ggt tcg gcg cga gtg acc 672
Gln Ser Phe Ser His Gln Thr Gln Trp Ser Gly Ser Ala Arg Val Thr
210 215 220
aaa aat ggc gaa atc aag ctg ttc ttc acc gac gtt gcg ttc tac cgc 720
Lys Asn Gly Glu Ile Lys Leu Phe Phe Thr Asp Val Ala Phe Tyr Arg
225 230 235 240
gac aag gac 729
Asp Lys Asp
<210> 8
<211> 243
<212> PRT
<213> Artificial
<220>
<223> Synthetic Construct
<400> 8
Met Glu Arg Ala Cys Val Ala Val Arg Glu Ile Val Arg Phe His Ile
1 5 10 15
Glu Gln Arg Gln Thr Ile Val Asn Lys Gln Arg Thr Lys Arg Gly Ile
20 25 30
Leu Ala Ala Ala Leu Ser Ile Gly Ala Leu Gly Ala Thr Leu Ile Ser
35 40 45
Gly Pro Ala Met Ala Ala Thr Glu Pro Val Pro Gly Phe Pro Gln Pro
50 55 60
Thr Glu His Thr Gln Lys Ala Tyr Ser Pro Thr Asp Asp Phe Thr Ser
65 70 75 80
Arg Trp Thr Arg Ala Asp Ala Lys Gln Ile Lys Ala Met Ser Asp Pro
85 90 95
Asn Ala Gly Ser Arg Glu Asn Ser Met Pro Lys Glu Tyr Thr Met Pro
100 105 110
Thr Val Pro Gln Asp Phe Pro Asp Met Ser Asn Glu Glu Val Trp Val
115 120 125
Trp Asp Thr Trp Pro Leu Thr Asp Glu His Ala Asn Gln Tyr Ser Val
130 135 140
Asn Gly Gln Glu Ile Ile Phe Ser Leu Val Ala Asp Arg Asp Leu Gly
145 150 155 160
Phe Asp Glu Arg His Gln Tyr Ala Arg Ile Gly Tyr Phe Tyr Arg Pro
165 170 175
Ala Gly Val Pro Ala Asp Glu Arg Pro Glu Asp Gly Gly Trp Thr Tyr
180 185 190
Gly Gly Gln Val Phe Asp Glu Gly Val Thr Gly Lys Ile Phe Glu Asp
195 200 205
Gln Ser Phe Ser His Gln Thr Gln Trp Ser Gly Ser Ala Arg Val Thr
210 215 220
Lys Asn Gly Glu Ile Lys Leu Phe Phe Thr Asp Val Ala Phe Tyr Arg
225 230 235 240
Asp Lys Asp
<210> 9
<211> 936
<212> DNA
<213> Artificial
<220>
<223> bff312
<220>
<221> CDS
<222> (1)..(936)
<400> 9
atg gag aga gcg tgt gtc gcg gtc cga gag att gtc cga ttc cat atc 48
Met Glu Arg Ala Cys Val Ala Val Arg Glu Ile Val Arg Phe His Ile
1 5 10 15
gaa caa agg caa aca atc gtg aac aag caa cgc acc aaa cgc ggc att 96
Glu Gln Arg Gln Thr Ile Val Asn Lys Gln Arg Thr Lys Arg Gly Ile
20 25 30
ctt gcc gca gca ttg agc atc gga gcg ctc gga gca act ttg att tcg 144
Leu Ala Ala Ala Leu Ser Ile Gly Ala Leu Gly Ala Thr Leu Ile Ser
35 40 45
ggg cca gcc atg gct gcc acc gaa cca gtg cct ggc ttc ccc cag cca 192
Gly Pro Ala Met Ala Ala Thr Glu Pro Val Pro Gly Phe Pro Gln Pro
50 55 60
acc gag cac acc cag aag gca tac agc ccc acc gac gat ttc acc tcg 240
Thr Glu His Thr Gln Lys Ala Tyr Ser Pro Thr Asp Asp Phe Thr Ser
65 70 75 80
cgt tgg acc cgt gct gac gca aag cag atc aag gcc atg tcc gac ccg 288
Arg Trp Thr Arg Ala Asp Ala Lys Gln Ile Lys Ala Met Ser Asp Pro
85 90 95
aac gcg gga tcg cgt gaa aac tcc atg ccc aag gag tac acc atg cct 336
Asn Ala Gly Ser Arg Glu Asn Ser Met Pro Lys Glu Tyr Thr Met Pro
100 105 110
acc gtg ccc cag gat ttc cct gac atg agc aac gag gaa gtc tgg gtt 384
Thr Val Pro Gln Asp Phe Pro Asp Met Ser Asn Glu Glu Val Trp Val
115 120 125
tgg gat act tgg cca ctg act gac gag cac gcc aac cag tac agc gtt 432
Trp Asp Thr Trp Pro Leu Thr Asp Glu His Ala Asn Gln Tyr Ser Val
130 135 140
aat ggc cag gag atc atc ttc tcg ctg gtt gcc gat cgc gac ctt ggc 480
Asn Gly Gln Glu Ile Ile Phe Ser Leu Val Ala Asp Arg Asp Leu Gly
145 150 155 160
ttc gat gag cgc cac cag tac gca cgc att gga tac ttc tac cgt cca 528
Phe Asp Glu Arg His Gln Tyr Ala Arg Ile Gly Tyr Phe Tyr Arg Pro
165 170 175
gca ggc gtt cct gcc gat gag cgt cct gaa gat ggt ggc tgg acc tat 576
Ala Gly Val Pro Ala Asp Glu Arg Pro Glu Asp Gly Gly Trp Thr Tyr
180 185 190
ggc ggc cag gtc ttc gat gag ggc gtc acc ggc aag atc ttc gag gac 624
Gly Gly Gln Val Phe Asp Glu Gly Val Thr Gly Lys Ile Phe Glu Asp
195 200 205
cag tcc ttc agc cac cag act cag tgg tcc ggt tcg gcg cga gtg acc 672
Gln Ser Phe Ser His Gln Thr Gln Trp Ser Gly Ser Ala Arg Val Thr
210 215 220
aaa aat ggc gaa atc aag ctg ttc ttc acc gac gtt gcg ttc tac cgc 720
Lys Asn Gly Glu Ile Lys Leu Phe Phe Thr Asp Val Ala Phe Tyr Arg
225 230 235 240
gac aag gac ggc aag gac atc aag cca tac gac ccg cgc atc gca ttg 768
Asp Lys Asp Gly Lys Asp Ile Lys Pro Tyr Asp Pro Arg Ile Ala Leu
245 250 255
agc gtt ggc cac gtc cac tcg aac aag aat ggc gtg aag ctc acc ggc 816
Ser Val Gly His Val His Ser Asn Lys Asn Gly Val Lys Leu Thr Gly
260 265 270
ttt gac aag gtc acc gac ctg ctg cag gca gac ggc aag tac tac cag 864
Phe Asp Lys Val Thr Asp Leu Leu Gln Ala Asp Gly Lys Tyr Tyr Gln
275 280 285
acc gct gag cag aac tcg tac ttc aac ttc cgt gat cca ttc acc ttt 912
Thr Ala Glu Gln Asn Ser Tyr Phe Asn Phe Arg Asp Pro Phe Thr Phe
290 295 300
gaa gat cca gcg cat cca ggc gaa 936
Glu Asp Pro Ala His Pro Gly Glu
305 310
<210> 10
<211> 312
<212> PRT
<213> Artificial
<220>
<223> Synthetic Construct
<400> 10
Met Glu Arg Ala Cys Val Ala Val Arg Glu Ile Val Arg Phe His Ile
1 5 10 15
Glu Gln Arg Gln Thr Ile Val Asn Lys Gln Arg Thr Lys Arg Gly Ile
20 25 30
Leu Ala Ala Ala Leu Ser Ile Gly Ala Leu Gly Ala Thr Leu Ile Ser
35 40 45
Gly Pro Ala Met Ala Ala Thr Glu Pro Val Pro Gly Phe Pro Gln Pro
50 55 60
Thr Glu His Thr Gln Lys Ala Tyr Ser Pro Thr Asp Asp Phe Thr Ser
65 70 75 80
Arg Trp Thr Arg Ala Asp Ala Lys Gln Ile Lys Ala Met Ser Asp Pro
85 90 95
Asn Ala Gly Ser Arg Glu Asn Ser Met Pro Lys Glu Tyr Thr Met Pro
100 105 110
Thr Val Pro Gln Asp Phe Pro Asp Met Ser Asn Glu Glu Val Trp Val
115 120 125
Trp Asp Thr Trp Pro Leu Thr Asp Glu His Ala Asn Gln Tyr Ser Val
130 135 140
Asn Gly Gln Glu Ile Ile Phe Ser Leu Val Ala Asp Arg Asp Leu Gly
145 150 155 160
Phe Asp Glu Arg His Gln Tyr Ala Arg Ile Gly Tyr Phe Tyr Arg Pro
165 170 175
Ala Gly Val Pro Ala Asp Glu Arg Pro Glu Asp Gly Gly Trp Thr Tyr
180 185 190
Gly Gly Gln Val Phe Asp Glu Gly Val Thr Gly Lys Ile Phe Glu Asp
195 200 205
Gln Ser Phe Ser His Gln Thr Gln Trp Ser Gly Ser Ala Arg Val Thr
210 215 220
Lys Asn Gly Glu Ile Lys Leu Phe Phe Thr Asp Val Ala Phe Tyr Arg
225 230 235 240
Asp Lys Asp Gly Lys Asp Ile Lys Pro Tyr Asp Pro Arg Ile Ala Leu
245 250 255
Ser Val Gly His Val His Ser Asn Lys Asn Gly Val Lys Leu Thr Gly
260 265 270
Phe Asp Lys Val Thr Asp Leu Leu Gln Ala Asp Gly Lys Tyr Tyr Gln
275 280 285
Thr Ala Glu Gln Asn Ser Tyr Phe Asn Phe Arg Asp Pro Phe Thr Phe
290 295 300
Glu Asp Pro Ala His Pro Gly Glu
305 310
<210> 11
<211> 1050
<212> DNA
<213> Artificial
<220>
<223> bff350
<220>
<221> CDS
<222> (1)..(1050)
<400> 11
atg gag aga gcg tgt gtc gcg gtc cga gag att gtc cga ttc cat atc 48
Met Glu Arg Ala Cys Val Ala Val Arg Glu Ile Val Arg Phe His Ile
1 5 10 15
gaa caa agg caa aca atc gtg aac aag caa cgc acc aaa cgc ggc att 96
Glu Gln Arg Gln Thr Ile Val Asn Lys Gln Arg Thr Lys Arg Gly Ile
20 25 30
ctt gcc gca gca ttg agc atc gga gcg ctc gga gca act ttg att tcg 144
Leu Ala Ala Ala Leu Ser Ile Gly Ala Leu Gly Ala Thr Leu Ile Ser
35 40 45
ggg cca gcc atg gct gcc acc gaa cca gtg cct ggc ttc ccc cag cca 192
Gly Pro Ala Met Ala Ala Thr Glu Pro Val Pro Gly Phe Pro Gln Pro
50 55 60
acc gag cac acc cag aag gca tac agc ccc acc gac gat ttc acc tcg 240
Thr Glu His Thr Gln Lys Ala Tyr Ser Pro Thr Asp Asp Phe Thr Ser
65 70 75 80
cgt tgg acc cgt gct gac gca aag cag atc aag gcc atg tcc gac ccg 288
Arg Trp Thr Arg Ala Asp Ala Lys Gln Ile Lys Ala Met Ser Asp Pro
85 90 95
aac gcg gga tcg cgt gaa aac tcc atg ccc aag gag tac acc atg cct 336
Asn Ala Gly Ser Arg Glu Asn Ser Met Pro Lys Glu Tyr Thr Met Pro
100 105 110
acc gtg ccc cag gat ttc cct gac atg agc aac gag gaa gtc tgg gtt 384
Thr Val Pro Gln Asp Phe Pro Asp Met Ser Asn Glu Glu Val Trp Val
115 120 125
tgg gat act tgg cca ctg act gac gag cac gcc aac cag tac agc gtt 432
Trp Asp Thr Trp Pro Leu Thr Asp Glu His Ala Asn Gln Tyr Ser Val
130 135 140
aat ggc cag gag atc atc ttc tcg ctg gtt gcc gat cgc gac ctt ggc 480
Asn Gly Gln Glu Ile Ile Phe Ser Leu Val Ala Asp Arg Asp Leu Gly
145 150 155 160
ttc gat gag cgc cac cag tac gca cgc att gga tac ttc tac cgt cca 528
Phe Asp Glu Arg His Gln Tyr Ala Arg Ile Gly Tyr Phe Tyr Arg Pro
165 170 175
gca ggc gtt cct gcc gat gag cgt cct gaa gat ggt ggc tgg acc tat 576
Ala Gly Val Pro Ala Asp Glu Arg Pro Glu Asp Gly Gly Trp Thr Tyr
180 185 190
ggc ggc cag gtc ttc gat gag ggc gtc acc ggc aag atc ttc gag gac 624
Gly Gly Gln Val Phe Asp Glu Gly Val Thr Gly Lys Ile Phe Glu Asp
195 200 205
cag tcc ttc agc cac cag act cag tgg tcc ggt tcg gcg cga gtg acc 672
Gln Ser Phe Ser His Gln Thr Gln Trp Ser Gly Ser Ala Arg Val Thr
210 215 220
aaa aat ggc gaa atc aag ctg ttc ttc acc gac gtt gcg ttc tac cgc 720
Lys Asn Gly Glu Ile Lys Leu Phe Phe Thr Asp Val Ala Phe Tyr Arg
225 230 235 240
gac aag gac ggc aag gac atc aag cca tac gac ccg cgc atc gca ttg 768
Asp Lys Asp Gly Lys Asp Ile Lys Pro Tyr Asp Pro Arg Ile Ala Leu
245 250 255
agc gtt ggc cac gtc cac tcg aac aag aat ggc gtg aag ctc acc ggc 816
Ser Val Gly His Val His Ser Asn Lys Asn Gly Val Lys Leu Thr Gly
260 265 270
ttt gac aag gtc acc gac ctg ctg cag gca gac ggc aag tac tac cag 864
Phe Asp Lys Val Thr Asp Leu Leu Gln Ala Asp Gly Lys Tyr Tyr Gln
275 280 285
acc gct gag cag aac tcg tac ttc aac ttc cgt gat cca ttc acc ttt 912
Thr Ala Glu Gln Asn Ser Tyr Phe Asn Phe Arg Asp Pro Phe Thr Phe
290 295 300
gaa gat cca gcg cat cca ggc gaa acc tac atg gtt ttc gag ggc aac 960
Glu Asp Pro Ala His Pro Gly Glu Thr Tyr Met Val Phe Glu Gly Asn
305 310 315 320
acc gct cag aag cgc gat gaa gcc aag tgc acc tcc gag gac ctt ggc 1008
Thr Ala Gln Lys Arg Asp Glu Ala Lys Cys Thr Ser Glu Asp Leu Gly
325 330 335
tac cgc aag ggt gaa acc aac ggc gaa acc gtg gat gag gtc 1050
Tyr Arg Lys Gly Glu Thr Asn Gly Glu Thr Val Asp Glu Val
340 345 350
<210> 12
<211> 350
<212> PRT
<213> Artificial
<220>
<223> Synthetic Construct
<400> 12
Met Glu Arg Ala Cys Val Ala Val Arg Glu Ile Val Arg Phe His Ile
1 5 10 15
Glu Gln Arg Gln Thr Ile Val Asn Lys Gln Arg Thr Lys Arg Gly Ile
20 25 30
Leu Ala Ala Ala Leu Ser Ile Gly Ala Leu Gly Ala Thr Leu Ile Ser
35 40 45
Gly Pro Ala Met Ala Ala Thr Glu Pro Val Pro Gly Phe Pro Gln Pro
50 55 60
Thr Glu His Thr Gln Lys Ala Tyr Ser Pro Thr Asp Asp Phe Thr Ser
65 70 75 80
Arg Trp Thr Arg Ala Asp Ala Lys Gln Ile Lys Ala Met Ser Asp Pro
85 90 95
Asn Ala Gly Ser Arg Glu Asn Ser Met Pro Lys Glu Tyr Thr Met Pro
100 105 110
Thr Val Pro Gln Asp Phe Pro Asp Met Ser Asn Glu Glu Val Trp Val
115 120 125
Trp Asp Thr Trp Pro Leu Thr Asp Glu His Ala Asn Gln Tyr Ser Val
130 135 140
Asn Gly Gln Glu Ile Ile Phe Ser Leu Val Ala Asp Arg Asp Leu Gly
145 150 155 160
Phe Asp Glu Arg His Gln Tyr Ala Arg Ile Gly Tyr Phe Tyr Arg Pro
165 170 175
Ala Gly Val Pro Ala Asp Glu Arg Pro Glu Asp Gly Gly Trp Thr Tyr
180 185 190
Gly Gly Gln Val Phe Asp Glu Gly Val Thr Gly Lys Ile Phe Glu Asp
195 200 205
Gln Ser Phe Ser His Gln Thr Gln Trp Ser Gly Ser Ala Arg Val Thr
210 215 220
Lys Asn Gly Glu Ile Lys Leu Phe Phe Thr Asp Val Ala Phe Tyr Arg
225 230 235 240
Asp Lys Asp Gly Lys Asp Ile Lys Pro Tyr Asp Pro Arg Ile Ala Leu
245 250 255
Ser Val Gly His Val His Ser Asn Lys Asn Gly Val Lys Leu Thr Gly
260 265 270
Phe Asp Lys Val Thr Asp Leu Leu Gln Ala Asp Gly Lys Tyr Tyr Gln
275 280 285
Thr Ala Glu Gln Asn Ser Tyr Phe Asn Phe Arg Asp Pro Phe Thr Phe
290 295 300
Glu Asp Pro Ala His Pro Gly Glu Thr Tyr Met Val Phe Glu Gly Asn
305 310 315 320
Thr Ala Gln Lys Arg Asp Glu Ala Lys Cys Thr Ser Glu Asp Leu Gly
325 330 335
Tyr Arg Lys Gly Glu Thr Asn Gly Glu Thr Val Asp Glu Val
340 345 350
Claims (8)
1.一种融合标签蛋白，其特征在于，所述融合标签蛋白基于耐有机糖苷酶Fru6构建，为截断至耐有机糖苷酶Fru6N端前n个氨基酸截短体，所述n＜452，所述耐有机糖苷酶Fru6氨基酸序列如序列表中SEQ ID No：1所示。
2.根据权利要求1所述融合标签蛋白，其特征在于，所述融合标签蛋白为截断至耐有机糖苷酶Fru6N端前209个氨基酸截短体，其氨基酸序列如序列表中SEQ ID No：4所示。
3.根据权利要求1所述融合标签蛋白，其特征在于，所述融合标签蛋白为截断至耐有机糖苷酶Fru6N端前217个氨基酸截短体，其氨基酸序列如序列表中SEQ ID No：6所示。
4.根据权利要求1所述融合标签蛋白，其特征在于，所述融合标签蛋白为截断至耐有机糖苷酶Fru6N端前243个氨基酸截短体，其氨基酸序列如序列表中SEQ ID No：8所示。
5.根据权利要求1所述融合标签蛋白，其特征在于，所述融合标签蛋白为截断至耐有机糖苷酶Fru6N端前312个氨基酸截短体，其氨基酸序列如序列表中SEQ ID No：10所示。
6.根据权利要求1所述融合标签蛋白，其特征在于，所述融合标签蛋白为截断至耐有机糖苷酶Fru6N端前350个氨基酸截短体，其氨基酸序列如序列表中SEQ ID No：12所示。
7.如权利要求1-6任一项所述融合标签蛋白的编码基因，以及含有该编码基因的重组载体，表达盒，转基因细胞系或重组菌。
8.如权利要求1-6任一项所述融合标签蛋白构建的融合蛋白表达系统。
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108588058A (zh) * | 2018-04-28 | 2018-09-28 | 南京工业大学 | β-呋喃果糖苷酶突变体及其应用 |
| CN113388009A (zh) * | 2021-05-18 | 2021-09-14 | 中国科学院福建物质结构研究所 | 一种标签蛋白及其编码基因、重组载体与应用 |
| CN117801124A (zh) * | 2024-02-29 | 2024-04-02 | 天津凯莱英生物科技有限公司 | 利西那肽前体的融合蛋白及其应用 |
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| US20120107876A1 (en) * | 2009-05-01 | 2012-05-03 | Lupin Limited | Novel fusion tag offering solubility to insoluble recombinant protein |
| CN103254277A (zh) * | 2013-05-03 | 2013-08-21 | 南京工业大学 | 强分泌性信号肽增强小肽模序及其应用 |
| CN103554245A (zh) * | 2013-10-15 | 2014-02-05 | 北京大学 | 一种融合标签蛋白 |
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| CN108588058A (zh) * | 2018-04-28 | 2018-09-28 | 南京工业大学 | β-呋喃果糖苷酶突变体及其应用 |
| CN108588058B (zh) * | 2018-04-28 | 2020-04-21 | 南京工业大学 | β-呋喃果糖苷酶突变体及其应用 |
| CN113388009A (zh) * | 2021-05-18 | 2021-09-14 | 中国科学院福建物质结构研究所 | 一种标签蛋白及其编码基因、重组载体与应用 |
| CN113388009B (zh) * | 2021-05-18 | 2022-07-19 | 中国科学院福建物质结构研究所 | 一种标签蛋白及其编码基因、重组载体与应用 |
| CN117801124A (zh) * | 2024-02-29 | 2024-04-02 | 天津凯莱英生物科技有限公司 | 利西那肽前体的融合蛋白及其应用 |
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| CN105838694B (zh) | 2019-07-26 |
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