CN110776572B - 一种7β-HSDH酶突变体及其制备方法 - Google Patents

一种7β-HSDH酶突变体及其制备方法 Download PDF

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CN110776572B
CN110776572B CN201911114498.3A CN201911114498A CN110776572B CN 110776572 B CN110776572 B CN 110776572B CN 201911114498 A CN201911114498 A CN 201911114498A CN 110776572 B CN110776572 B CN 110776572B
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王峰
吕志佳
徐霞
陈倩
缪奇微
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Wuxi Baiaode Biological Science Co.,Ltd.
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Abstract

本发明涉及一种7β‑HSDH酶与DPS融合蛋白，将氨基酸序列如SEQUENCE NO.1所示的7β‑HSDH酶的第159位甘氨酸突变成组氨酸，第165位赖氨酸突变成组氨酸，得到氨基酸序列如SEQUENCE NO.2所示的7β‑HSDH酶突变体，将7β‑HSDH酶突变体与来源于Thermoanaerobacter pseudethanolicus种属的DPS蛋白进行融合表达得到7β‑HSDH酶与DPS融合蛋白。与野生7β‑HSDH酶相比，本发明所得的7β‑HSDH酶与DPS融合蛋白的热稳定性显著提高，酶活性提高了70％。

Description

一种7β-HSDH酶突变体及其制备方法
技术领域
本发明涉及融合蛋白技术领域，具体地，涉及一种7β-HSDH酶与DPS融合蛋白。
背景技术
牛磺熊去氧胆酸(Tauroursodeoxycholic acid,TUDCA Ursodeoxycholic acid)是熊胆中的主要药理活性成分，目前主要利用人工提取熊胆汁的方式来获得。牛磺熊去氧胆酸在临床上主要用于治疗与胆汁酸代谢异常相关的疾病，但由于熊胆资源的稀缺性和来源的特殊性，使牛磺熊去氧胆酸(TUDCA)的产量远远不能满足临床用药需求。目前TUDCA除可以从熊胆中提取外，其制备的方法还有化学合成法和生物转化法。化学合成方法制备牛磺熊去氧胆酸(TUDCA)过程复杂且产量低，生物转化法中7β-HSDH酶的活性及热稳定性需要进一步提高，以满足工业生产的需求。
发明内容
本发明分析比较了来源于Ruminococcus torques、Ruminococcus gnavus、Collinsella aerofaciens、Clostridium sardiniense种属的4种7β-HSDH酶的热稳定性及酶活性，将来源于Ruminococcus gnavus种属的7β-HSDH酶进行点突变设计，将7β-HSDH酶突变体与DPS蛋白进行融合表达，以提高7β-HSDH酶的活性及热稳定性，方便后续工业催化应用。
一种7β-HSDH酶突变体，所述7β-HSDH酶突变体的氨基酸序列如SEQUENCE ID NO.2所示。
所述7β-HSDH酶突变体的制备方法为：对氨基酸序列如SEQUENCE NO.1所示的7β-HSDH酶的第159位甘氨酸突变成组氨酸，第165位赖氨酸突变成组氨酸，得到氨基酸序列如SEQUENCE NO.2所示的7β-HSDH酶突变体。
所述7β-HSDH酶来源于Ruminococcus gnavus种属。
将7β-HSDH酶突变体与来源于Thermoanaerobacter pseudethanolicus种属的DPS蛋白进行融合表达得到7β-HSDH酶与DPS融合蛋白。
所述的7β-HSDH酶与DPS融合蛋白的制备方法，通过PCR或基因合成的方法将7β-HSDH酶突变体与DPS蛋白构建到大肠杆菌pGEX6P-1载体上，获得的重组载体转化大肠杆菌BL21(DE3)感受态细胞，诱导表达重组蛋白，获得7β-HSDH酶与DPS融合蛋白。
与野生7β-HSDH酶相比，本发明所得的7β-HSDH与DPS融合蛋白的热稳定性和酶活性显著高于活性，酶活性提高了70％。
附图说明：
图1为pGEX6P-1-N-GST-3C-7β-HSDH突变体质粒图谱。
图2为pGEX6P-1-N-GST-3C-DPS-linker-7β-HSDH质粒图谱。
图3为7β-HSDH酶(Ruminococcus torques)SDS-PAGE电泳分析结果；泳道1：2ug目的蛋白，泳道2：10ug目的蛋白。
图4是7β-HSDH(Ruminococcus gnavus)SDS-PAGE电泳分析结果；泳道1：2ug目的蛋白，泳道2：10ug目的蛋白。
图5是7β-HSDH酶(Collinsella aerofaciens)SDS-PAGE电泳分析结果；泳道1：2ug目的蛋白，泳道2：10ug目的蛋白。
图6是7β-HSDH酶(Clostridium sardiniense)SDS-PAGE电泳分析结果；泳道1：2ug目的蛋白，泳道2：10ug目的蛋白。
图7是7β-HSDH酶与DPS融合蛋白SDS-PAGE电泳分析结果；泳道1：2ug目的蛋白，泳道2：10ug目的蛋白。
图8是4种种属的7β-HSDH酶以及7β-HSDH酶与DPS融合蛋白热稳定性分析结果。来源于Ruminococcus torques 7β-HSDH酶为图中1#曲线，来源于Ruminococcus gnavus 7β-HSDH酶为图中2#曲线，来源于Collinsella aerofaciens 7β-HSDH酶为图中3#曲线，来源于Clostridium sardiniense 7β-HSDH酶为图中4#曲线，7β-HSDH酶与DPS融合蛋白为图中5#曲线。
图9是4种种属的7β-HSDH酶以及7β-HSDH酶与DPS融合蛋白酶活性分析结果。
具体实施方式
下面通过具体的实施方案叙述本发明。除非特别说明，本发明所述的技术手段均为本领域技术人员所知的方法。此外，实施方案应理解为说明性的，而非限制本发明的范围。本发明的实质和范围仅由权利要求书所限定。
一、材料
1.1表达载体：(1)pGEX6P-1-N-GST-3C-7β-HSDH，其质粒图谱图如1所示；
(2)pGEX6P-1-N-GST-3C-DPS-linker-7β-HSDH由本公司自行构建，其质粒图谱图如2所示。
1.2感受态细胞：TransT1感受态细胞，BL21感受态细胞。
1.3培养基及试剂：LB培养基、IPTG诱导剂、L-Glutathione(Reduced)、SYPROOrange染料、NADP、TUDCA、质粒小提试剂盒(TIANGEN)、普通琼脂糖凝胶回收试剂盒(TIANGEN)
1.4实验设备SPH-211C控温摇床、AKTA-FPLC快速蛋白纯化液相、安捷伦LC-MS质谱分析仪、Biometra TProfessional PCR仪、LRH-70F生化培养箱、MD SpectraMax Plus384连续波长酶标仪、ABI7500.
二、方法
2.1 7β-HSDH酶点突变设计
通过PCR或基因合成的方法分别获得源于Ruminococcus torques、Ruminococcusgnavus、Collinsella aerofaciens、Clostridium sardiniense种属的4种野生型7β-羟基类固醇脱氢酶(7β-HSDH)的全基因序列DNA。
源于Ruminococcus gnavus种属的7β-HSDH氨基酸序列，如SEQUENCE NO.1所示。对源于Ruminococcus gnavus种属的7β-HSDH全基因序列进行点突变设计，将第159位甘氨酸突变成组氨酸，第165位赖氨酸突变成组氨酸。通过定点突变方法，获得7β-HSDH酶(G159H，K165H)突变序列,如SEQUENCE NO.2所示。
2.2 7β-HSDH表达载体构建
以GST作为亲和标签，利用PCR、无缝克隆方法，将源于Ruminococcus gnavus种属的7β-HSDH酶(G159H，K165H)突变序列和来源于Thermoanaerobacter pseudethanolicus种属的DPS蛋白基因导入大肠杆菌pGEX6P-1载体，构建重组质粒：pGEX6P-1-N-GST-3C-7β-HSDH和pGEX6P-1-N-GST-3C-DPS-linker-7β-HSDH。
同时，将源于Ruminococcus torques、Ruminococcus gnavus、Collinsellaaerofaciens、Clostridium sardiniense种属的4种野生型7β-羟基类固醇脱氢酶(7β-HSDH)的全基因序列DNA导入大肠杆菌pGEX6P-1载体，构建重组质粒：pGEX6P-1-N-GST-3C-7β-HSDH
2.3 7β-HSDH蛋白表达
准备大肠杆菌BL21(DE3)感受态，将重组质粒：pGEX6P-1-N-GST-3C-7β-HSDH和pGEX6P-1-N-GST-3C-DPS-linker-7β-HSDH转化到大肠杆菌BL21(DE3)感受态，按照培养温度15℃，IPTG终浓度0.5mM，诱导时间16h，从2L液体培养基中获得目的蛋白。
2.4 7β-HSDH酶与DPS融合蛋白纯化及SDS-PAGE电泳分析
使用亲和层析法对4种7β-HSDH酶、7β-HSDH酶突变体与DPS融合蛋白进行纯化，并利用SDS-PAGE电泳分析4种7β-HSDH酶、7β-HSDH酶突变体与DPS融合蛋白表达及纯化效果。如图3，图4，图5，图6和图7所示。
2.5 7β-HSDH酶与DPS融合蛋白热稳定性分析
利用SYPRO Orange染料对7β-HSDH酶、7β-HSDH酶与DPS融合蛋白进行热稳定性分析，在96孔微孔板中加入蛋白纯化缓冲液、目的蛋白以及SYPRO Orange染料。程序设置为：95℃2min，25℃2min，温度上升速度设定为每分钟1℃。如图8所示。
2.6 7β-HSDH酶与DPS融合蛋白酶活性分析
对获得的7β-HSDH酶、7β-HSDH酶与DPS融合蛋白进行酶活性分析，在96孔反应板中加入缓冲液、目的蛋白、NADP以及TUDCA，测定340nM处吸光值的变化。
三、结果分析
3.1蛋白表达量分析
经实验测定，四种7β-HSDH酶以及7β-HSDH酶突变体与DPS融合蛋白在相同的表达条件下，各蛋白的表达量存在差异，7β-HSDH酶与DPS融合蛋白表达量最高，来源于Ruminococcus gnavus种属的7β-HSDH酶表达量次之，见表1。
3.2热稳定性结果分析
经实验测定，7β-HSDH酶与DPS融合蛋白的Tm平均值为64.43℃，来源于Ruminococcus torques种属的7β-HSDH酶Tm平均值为55.01，来源于Ruminococcus gnavus种属的7β-HSDH酶Tm平均值为55.85，来源于Collinsella aerofaciens种属的7β-HSDH酶Tm平均值为54.58，来源于Clostridium sardiniense种属的7β-HSDH酶Tm平均值为54.07，7β-HSDH酶与DPS融合蛋白稳定性比4种种属的7β-HSDH酶的热稳定性提高了20％，见表1。
3.3酶活性结果分析
经实验测定，来源于Ruminococcus torques种属的7β-HSDH酶活性为41.1U/mg，来源于Ruminococcus gnavus种属的7β-HSDH酶活性为68.8U/mg，来源于Collinsellaaerofaciens种属的7β-HSDH酶活性30.5U/mg，来源于Clostridium sardiniense种属的7β-HSDH酶活性为34.1U/mg，7β-HSDH酶与DPS融合蛋白酶活性为120.8U/mg，7β-HSDH酶与DPS融合蛋白酶活性显著高于4种种属的7β-HSDH酶活性，酶活性提高了70％。见表1和图9。
表1各蛋白表达量、热稳定性、酶活性比较
Figure GDA0003069715670000051
以上内容是结合具体的优选实施方式对本发明所作的进一步详细说明，不能认定本发明的具体实施只局限于这些说明。对于本发明所属技术领域的普通技术人员来说，在不脱离本发明构思的前提下，其架构形式能够灵活多变，可以派生系列产品。只是做出若干简单推演或替换，都应当视为属于本发明由所提交的权利要求书确定的专利保护范围。
序列表
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Claims (3)

1.一种7β-HSDH酶突变体，其特征在于，所述7β-HSDH酶突变体的氨基酸序列如SEQUENCE ID NO.2所示。
2.一种如权利要求1所述7β-HSDH酶突变体的制备方法，其特征在于，对氨基酸序列如SEQUENCE NO.1所示的7β-HSDH酶的第159位甘氨酸突变成组氨酸，第165位赖氨酸突变成组氨酸，得到氨基酸序列如SEQUENCE ID NO.2所示的7β-HSDH酶突变体。
3.根据权利要求2所述的7β-HSDH酶突变体的制备方法，其特征在于，所述7β-HSDH酶来源于Ruminococcus gnavus种属。
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