CN114958790B - 肝素骨架合酶及其突变体与应用 - Google Patents
肝素骨架合酶及其突变体与应用 Download PDFInfo
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- CN114958790B CN114958790B CN202110192424.2A CN202110192424A CN114958790B CN 114958790 B CN114958790 B CN 114958790B CN 202110192424 A CN202110192424 A CN 202110192424A CN 114958790 B CN114958790 B CN 114958790B
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Abstract
本发明涉及一种肝素骨架合酶及其突变体与应用。本发明中的肝素骨架合酶来源于动物奈瑟菌,其氨基酸序列如SEQ ID NO.2所示,编码基因的核苷酸序列如SEQ ID NO.1所示,重组表达水平是现有大肠杆菌K5肝素骨架合酶KfiA重组表达水平的6.8倍,每升发酵液的总酶活是肝素骨架合酶KfiA的5.22倍。对上述肝素骨架合酶氨基酸序列的第16位、25位、30位、111位、165位、172位进行定点突变,所得肝素骨架合酶突变体均具有较高的表达水平。本发明中的肝素骨架合酶及其突变体提高了肝素骨架合成中GlcNAc转移合成效率,极大地促进了肝素仿生合成应用化发展,为糖胺聚糖的研究发展开启了全新的一页。
Description
技术领域
本发明涉及一种肝素骨架合酶及其突变体与应用,具体涉及来源于动物奈瑟菌(Neisseria animaloris)的肝素骨架合酶及其六种突变体与在肝素骨架合成中的应用,属于生物技术领域。
背景技术
肝素(Heparin,简称HP),是一种重要的糖胺聚糖,是由D-β-葡糖醛酸(或L-α-艾杜糖醛酸)和N-乙酰氨基葡糖形成重复二糖单位所构成的。肝素有着巨大的药用价值,除抗凝作用等多种运用外,还可用于治疗心绞痛、肾病综合征、严重烧伤、类风湿性关节炎等,其需求量在国际上常年处于生物技术药物领域的前列。
肝素的生产主要是从牛肺、猪肠粘膜等动物组织器官中提取获得,然而天然提取的方法安全性低,得率低,溶剂的消耗量大,环境污染严重,并且产品质量由于原材料差异大不易控制,在生产过程中容易引入杂质污染(如硫酸软骨素)。天然肝素基本单元的结构、数目各不相同,提取方法的不同也会导致不同的化学修饰,导致最后得到的肝素产物结构、构型、分子量各有异同,最终引起最终产物活性不均一,并且难以进行完善的质量监控,使肝素的品质和安全性都难以得到保障。
2008年爆发的肝素大面积污染事件,造成近100位病人死亡,该事件的发生极大地促进了非动物来源肝素生产的发展。化学酶法合成策略因具有立体选择性强,产率高,反应温和,产品质量均一稳定,容易进行官能团的衍生和修饰,对于新药研制具有重要意义,有望发展成为一种理想的肝素寡糖的合成新技术。化学酶法所使用工具酶是推动该策略进一步发展的制约步骤。
目前为止,已确认具有N-乙酰氨基葡糖转移酶活性的微生物来源的肝素骨架合酶(N-Acetyl-D-glucosaminyltransferase)已有2种被报道,分别是来源于大肠杆菌K5中的KfiA和来自卡氏杆菌中的GaKfiA。肝素骨架合酶的匮乏不但限制了该酶家族酶学特性的理论研究,也限制了化学酶法合成肝素体系的规模化应用。
动物奈瑟菌(Neisseria animaloris)是一种罕见的人畜共患病病原体,通常与狗或猫咬伤有关。动物奈瑟菌主要存在于犬类和猫科动物口腔,在被咬伤后会在人类和动物身上引起全身感染。搜索数据库并未见来源于该菌的糖胺聚糖骨架合酶基因被报道。
发明内容
针对现有技术的不足,本发明提供了一种肝素骨架合酶及其突变体与应用,本发明中的肝素骨架合酶为来源于动物奈瑟菌(Neisseria animaloris)的高表达水平的肝素骨架合酶NaGlcNAc-T,并经定点突变得到六种高活性突变体,该肝素骨架合酶及六种高活性突变体可应用于肝素寡糖的合成。
术语说明:
GlcA-pNP:中文全称为4-硝基苯基-β-D-葡萄糖醛酸,其作用是作为肝素寡糖合成的起始底物;
UDP-GlcNAc:中文全称为尿苷二磷酸-N-乙酰氨基葡萄糖,其作用是为肝素寡糖的合成提供乙酰葡萄糖胺供体;
UDP-GlcNTFA:中文全称为尿苷二磷酸-N-三氟乙酰氨基葡萄糖,其作用是测定肝素骨架合酶的底物特异性,提供三氟乙酰氨基葡萄糖供体;
UDP-GalNAz:中文全称为尿苷二磷酸-N-叠氮乙酰氨基半乳糖,其作用是测定肝素骨架合酶的底物特异性,提供叠氮乙酰氨基半乳糖供体;
UDP-GalNAc:中文全称为尿苷二磷酸-N-乙酰氨基半乳糖,其作用是测定肝素骨架合酶的底物特异性,提供乙酰氨基半乳糖供体;
UDP-Glc:中文全称为尿苷二磷酸-N-葡萄糖,其作用是测定肝素骨架合酶的底物特异性提供葡萄糖供体;
UDP-Gal:中文全称为尿苷二磷酸-N-半乳糖,其作用是测定肝素骨架合酶的底物特异性,提供半乳糖供体。
本发明技术方案如下:
一种肝素骨架合酶NaGlcNAc-T,其氨基酸序列如SEQ ID NO.2所示,编码基因的核苷酸序列如SEQ ID NO.1所示。
本发明中,所述肝素骨架合酶NaGlcNAc-T来源于动物奈瑟菌(Neisseriaanimaloris),重组表达水平是现有大肠杆菌K5肝素骨架合酶KfiA重组表达水平的6.8倍。对上述肝素骨架合酶NaGlcNAc-T进行定点突变,所得肝素骨架合酶突变体均具有较高的表达水平。
一种肝素骨架合酶突变体NaGlcNAc-T(C16L),其氨基酸序列如SEQ ID NO.4所示,编码基因的核苷酸序列如SEQ ID NO.3所示;是将上述肝素骨架合酶氨基酸序列中第16位的半胱氨酸定点突变为亮氨酸。
一种肝素骨架合酶突变体NaGlcNAc-T(N25P),其氨基酸序列如SEQ ID NO.6所示,编码基因的核苷酸序列如SEQ ID NO.5所示;是将上述肝素骨架合酶氨基酸序列中第25位的天冬酰胺定点突变为脯氨酸。
一种肝素骨架合酶突变体NaGlcNAc-T(I30L),其氨基酸序列如SEQ ID NO.8所示,编码基因的核苷酸序列如SEQ ID NO.7所示;是将上述肝素骨架合酶氨基酸序列中第30位的异亮氨酸定点突变为亮氨酸。
一种肝素骨架合酶突变体NaGlcNAc-T(I111S),其氨基酸序列如SEQ ID NO.10所示,编码基因的核苷酸序列如SEQ ID NO.9所示;是将上述肝素骨架合酶氨基酸序列中第111位的异亮氨酸定点突变为丝氨酸。
一种肝素骨架合酶突变体NaGlcNAc-T(S165K),其氨基酸序列如SEQ ID NO.12所示,编码基因的核苷酸序列如SEQ ID NO.11所示;是将上述肝素骨架合酶氨基酸序列中第165位的丝氨酸定点突变为赖氨酸。
一种肝素骨架合酶突变体NaGlcNAc-T(S172A),其氨基酸序列如SEQ ID NO.14所示,编码基因的核苷酸序列如SEQ ID NO.13所示;是将上述肝素骨架合酶氨基酸序列中第172位的丝氨酸定点突变为丙氨酸。
本发明中,所述肝素骨架合酶突变体也有较高的重组表达水平,其酶活性相对于肝素骨架合酶NaGlcNAc-T保持平稳或有不同程度的提升。
一种重组载体,是在质粒载体中插入上述肝素骨架合酶NaGlcNAc-T或上述肝素骨架合酶突变体的编码基因。
根据本发明优选的,所述质粒载体为pET30a(+)。
本发明中,含有目的基因的重组载体由南京金斯瑞公司合成。
一种重组菌株,是将上述重组载体转化入宿主细胞中获得。
根据本发明优选的,所述宿主细胞为大肠杆菌;进一步优选为含有pGro7质粒的大肠杆菌BL21(DE3)。
上述肝素骨架合酶NaGlcNAc-T或上述肝素骨架合酶突变体在合成肝素糖链中的应用。
根据本发明优选的,所述应用是以GlcA-pNP作为起始受体,UDP-GlcNAc作为供体,生成结构为GlcNAc-GlcA-pNP的肝素骨架二糖。
有益效果:
本发明公开的肝素骨架合酶NaGlcNAc-T,是一种全新来源的肝素骨架合酶,具备GlcNAc的转移酶活性。并且在最适条件下利用底物GlcA-pNP和UDP-GlcNAc有效地合成肝素二糖骨架。并经过初步表达分析,NaGlcNAc-T在每升普通的LB培养基可以表达超过100mg的可溶活性蛋白,相比已经投入应用的大肠杆菌K5肝素骨架合酶KfiA,每升发酵液表达的酶活提高了5.22倍。并且本发明设计突变得到了比KfiA活性更高的NaGlcNAc-T突变体。本发明提高了肝素骨架合成中GlcNAc转移合成效率,极大地促进了肝素仿生合成应用化发展,为糖胺聚糖的研究发展开启了全新的一页。
附图说明
图1为肝素骨架合酶NaGlcNAc-T及其突变体在重组大肠杆菌中可溶性表达纯化的SDS-PAGE检测结果图;
图中:M为marker;其余各条带为纯化得到的NaGlcNAc-T及其突变体的重组蛋白,NaKfiA为NaGlcNAc-T,C16L为NaGlcNAc-T(C16L),N25P为NaGlcNAc-T(N25P),I30L为NaGlcNAc-T(I30L),I111S为NaGlcNAc-T(I111S),S165K为NaGlcNAc-T(S165K),S172A为NaGlcNAc-T(S172A);
图2为肝素骨架合酶NaGlcNAc-T的蛋白定量标准曲线;
图3为肝素骨架合酶NaGlcNAc-T与肝素骨架合酶KfiA的表达量及受体底物反应转化率柱状图;
图4为肝素骨架合酶NaGlcNAc-T反应产物的HPLC色谱图;
图5为肝素骨架合酶NaGlcNAc-T与肝素骨架合酶KfiA的总酶活柱状图;
图6为肝素骨架合酶NaGlcNAc-T及其突变体的受体底物反应转化率柱状图;
图7为肝素骨架合酶NaGlcNAc-T反应产物GlcNAc-GlcA-pNP的质谱图;
图8为肝素骨架合酶NaGlcNAc-T反应产物GlcNAc-GlcA-pNP的1H-H COSY氢谱;
图9为不同pH下肝素骨架合酶NaGlcNAc-T催化体外寡糖合成时的受体底物反应转化率曲线;
图10为不同金属离子下肝素骨架合酶NaGlcNAc-T催化体外寡糖合成时受体底物反应转化率柱状图;
图11为不同温度下肝素骨架合酶NaGlcNAc-T体外催化寡糖合成时受体底物反应转化率曲线;
图12为不同供体底物下肝素骨架合酶NaGlcNAc-T体外催化寡糖合成时受体底物反应转化率柱状图;
图中:N.D.代表该底物不能被NaGlcNAc-T催化。
具体实施方式
下面结合实施例及说明书附图,对本发明的技术方案做进一步说明,但本发明所保护范围不限于此。除非特殊说明,本发明中所运用的技术手段均为本领域技术人员所公知的方法。
发明人通过使用Blast序列比对进行生物信息学数据库搜索,发现动物奈瑟菌(Neisseria animaloris,ATCC29858)中一段基因形成的氨基酸序列与之前已经报道的大肠杆菌K5肝素骨架合酶KfiA的氨基酸序列有55.7%的同源性,因此推测这段基因表达的蛋白产物可能具有肝素骨架合酶活性,在大肠杆菌表达系统中表达该蛋白,并将这段基因命名为NaGlcNAc-T,核苷酸序列如SEQ ID NO.1所示,将其表达的蛋白产物命名为NaGlcNAc-T,氨基酸序列如SEQ ID NO.2所示。
同时,发明人对于检索到的KfiA同源序列,进行序列同源分析,使用EMBL ClustalOmega工具进行多序列比对,并使用Jalview软件对于氨基酸序列高保守区域进行分析。同时使用Swiss-Model工具对NaGlcNAc-T进行蛋白质模拟建模,并使用HotSpot Wizard 2.0对该酶的活性中心进行预测。发现NaGlcNAc-T的氨基酸序列的16位、25位、30位、111位、165位、172位,位于活性中心及高保守区域附近,对其进行定点突变极有可能提高NaGlcNAc-T的催化活性。因此结合同源分析的在该六个位置的优势氨基酸,设计了NaGlcNAc-T(C16L)、NaGlcNAc-T(N25P)、NaGlcNAc-T(I30L)、NaGlcNAc-T(I111S)、NaGlcNAc-T(S165K)、NaGlcNAc-T(S172A)共计六个突变体,其中突变体NaGlcNAc-T(C16L)的核苷酸序列如SEQID NO.3所示,氨基酸序列如SEQ ID NO.4所示;突变体NaGlcNAc-T(N25P)的核苷酸序列如SEQ ID NO.5所示,氨基酸序列如SEQ ID NO.6所示;突变体NaGlcNAc-T(I30L)的核苷酸序列如SEQ ID NO.7所示,氨基酸序列如SEQ ID NO.8所示;突变体NaGlcNAc-T(I111S)的核苷酸序列如SEQ ID NO.9所示,氨基酸序列如SEQ ID NO.10所示;突变体NaGlcNAc-T(S165K)的核苷酸序列如SEQ ID NO.11所示,氨基酸序列如SEQ ID NO.12所示;突变体NaGlcNAc-T(S172A)的核苷酸序列如SEQ ID NO.13所示,氨基酸序列如SEQ ID NO.14所示。
本发明所采用的底物糖类试剂均购自于Sigma公司。所有的NaGlcNAc-T及其突变体的质粒均委托南京金斯瑞公司构建。使用的含有pGro7分子伴侣的BL21(DE3)感受态细胞购买自Takara公司。所采用的HPLC检测方法均使用YMC的氨基柱,液相系统为日本岛津液相,紫外检测系统为SPD-20A。检测NaGlcNAc-T及其突变体催化产物通过色谱柱分离后各组分在310nm和254nm的紫外吸收,HPLC流动相条件见表1:
表1.检测肝素寡糖所使用HPLC分析方法
实施例1.肝素骨架合酶NaGlcNAc-T及其突变体的重组蛋白的表达和纯化
(1)表达菌株的构建
将南京金斯瑞公司合成的重组质粒pET30a(+)-NaGlcNAc-T转化入含有pGro7分子伴侣的大肠杆菌BL21(DE3)感受态细胞中,在含卡那霉素(100μg/mL)和氯霉素(37μg/mL)的LB平板上培养12h,筛选转化子(同时进行阴性对照实验),获得阳性转化子。
按照上述相同方法得到六个突变体NaGlcNAc-T(C16L)、NaGlcNAc-T(N25P)、NaGlcNAc-T(I30L)、NaGlcNAc-T(I111S)、NaGlcNAc-T(S165K)、NaGlcNAc-T(S172A)的阳性转化子。
(2)NaGlcNAc-T及其突变体重组蛋白的表达和纯化
挑取NaGlcNAc-T阳性转化子单菌落于30mL灭菌的LB培养基(含有100μg/mL卡那霉素和37μg/mL氯霉素)中,进行活化培养(37℃,225r/min);将过夜活化培养的菌液按照1%接种量接入1L LB培养基(含有100μg/mL卡那霉素和37μg/mL氯霉素)中扩大培养,37℃,225r/min震荡培养大约4个小时至OD600约为0.8,加入终浓度0.5mM IPTG和1mg/mL L-阿拉伯糖,于22℃、225r/min条件下诱导16-18h,然后收集菌体,并用平衡缓冲液(20mM Tris-HCl,pH=8.00;0.5M NaCl;10mM咪唑)重悬,冰上超声破碎(工作3s,间歇5s,振幅33%,能量1500KJ,4℃)30min,将破碎后菌体12000rpm离心20min(4℃),上清用0.22μm滤膜过滤,运用镍柱进行纯化,上样后用平衡缓冲液洗涤,然后用洗杂缓冲液(20mM Tris-HCl,pH=8.00;0.5M NaCl;40mM咪唑)洗掉多余的杂蛋白,最后用洗脱缓冲液(20mM Tris-HCl,pH=8.00;0.5M NaCl;250mM咪唑)洗脱得到目的蛋白。纯化得到的蛋白质保存在20%甘油,每管分装存于-80℃冰箱。
按照上述相同方法纯化得到六个突变体NaGlcNAc-T(C16L)、NaGlcNAc-T(N25P)、NaGlcNAc-T(I30L)、NaGlcNAc-T(I111S)、NaGlcNAc-T(S165K)、NaGlcNAc-T(S172A)的重组蛋白。
通过聚丙烯酰胺凝胶电泳(SDS-PAGE)对纯化后的NaGlcNAc-T及其突变体重组蛋白进行鉴定(如图1所示)。结果显示NaGlcNAc-T及其突变体NaGlcNAc-T(C16L)、NaGlcNAc-T(N25P)、NaGlcNAc-T(I30L)、NaGlcNAc-T(I111S)、NaGlcNAc-T(S165K)、NaGlcNAc-T(S172A)均成功使用此法纯化得到,且相对纯度都达到了90%以上。
使用BCA蛋白浓度测定试剂盒(碧云天P0011)对NaGlcNAc-T进行定量。按50体积BCA试剂A加1体积BCA试剂B(50:1)配制适量BCA工作液,充分混匀。将标准品按0、4、8、12、16、20μL加到96孔板的标准品孔中,加标准品稀释液补足到20μL,相当于标准品浓度为0、0.1、0.2、0.3、0.4、0.5mg/mL。加20μL样品到96孔板的样品孔中。各孔加入200μL BCA工作液,37℃放置20-30分钟,用酶标仪测定A562,测得标准曲线,并根据标准曲线和使用的样品体积计算出样品的蛋白浓度。
NaGlcNAc-T蛋白定量的标准曲线如图2所示。经测定,NaGlcNAc-T在每升LB培养基的重组表达量高达102mg/L,这是采用同样方式进行重组表达的大肠杆菌K5肝素骨架合酶KfiA的蛋白表达量(~15mg/L)的6.8倍(如图3所示),这表明NaGlcNAc-T具有高表达水平,具有巨大的产业化应用前景。
按照上述相同方法测定六个突变体NaGlcNAc-T(C16L)、NaGlcNAc-T(N25P)、NaGlcNAc-T(I30L)、NaGlcNAc-T(I111S)、NaGlcNAc-T(S165K)、NaGlcNAc-T(S172A)的表达水平,发现上述六个突变体相对于大肠杆菌K5肝素骨架合酶KfiA,也具有较高的表达水平。
实施例2.肝素骨架合酶NaGlcNAc-T及其突变体活性的验证
(1)肝素骨架合酶NaGlcNAc-T的GlcNAc转移酶活性验证
用商业化GlcA-pNP(终浓度0.2mM)作为受体底物,UDP-GlcNAc(终浓度0.3mM)作为供体底物进行反应,反应体系如表2所示;该反应置于37℃水浴锅中反应4h,沸水加热5min使酶失活从而终止反应,反应液用0.22μm的滤膜过滤后按照表1所述方法进行HPLC检测,单糖受体的pNP基团在紫外310nm检测波长下有特异性吸收,流动相流速为0.5mL/min。
使用同样的方法检测六个突变体NaGlcNAc-T(C16L)、NaGlcNAc-T(N25P)、NaGlcNAc-T(I30L)、NaGlcNAc-T(I111S)、NaGlcNAc-T(S165K)、NaGlcNAc-T(S172A)的GlcNAc转移酶活性。
表2.NaGlcNAc-T及其突变体的GlcNAc转移酶活性验证反应体系
检测结果如图4所示,NaGlcNAc-T及各个突变体均具有GlcNAc转移酶活性,能将GlcNAc基团转移到GlcA-pNP的非还原端,生成肝素二糖GlcNAc-GlcA-pNP。其中,受体底物GlcA-pNP的反应转化率如图3所示,由反应转化率及蛋白酶的表达量计算得到蛋白酶的总酶活,结果如图5所示,NaGlcNAc-T的每升发酵液的总酶活是大肠杆菌K5肝素骨架合酶KfiA的5.22倍。
按照表2的反应体系及上述处理方法对六个突变体的活性进行测定,37℃水浴锅中反应1h,各个突变体的受体底物GlcA-pNP的反应转化率如图6所示,结果显示各个突变体相对于NaGlcNAc-T的活性均有不同程度的保持和提升,其中突变体NaGlcNAc-T(C16L)和突变体NaGlcNAc-T(S165K)活性最佳,这些突变体是高效催化肝素糖链合成的工具酶。
(2)肝素二糖GlcNAc-GlcA-pNP的质谱确证
为了确认上述活性反应的产物结构为GlcNAc-GlcA-pNP,随后进行电喷雾电离质谱(ESI-MS)分析。反应以较大规模进行以获得足够的二糖产物。产物通过P2柱进行纯化,然后在Thermo LCQ-Deca上进行MS分析。MS的所有样品都是通过溶解在50%甲醇中制备的。MS实验在负离子模式下进行,喷雾电压为5kV,毛细管温度为275℃。
质谱分析结果如图7所示,MS图谱中得到了与GlcNAc-GlcA-pNP(Mw=518.14)脱去一个质子后分子量相符合的峰(517.02),并且其二倍峰(1034.72)同时被检测到,证明产物GlcNAc-GlcA-pNP的生成。
(3)肝素二糖GlcNAc-GlcA-pNP的核磁确证
取约1mg上述活性反应产物GlcNAc-GlcA-pNP溶于500μL的重水中,使用600M核磁收集1H-NMR。如图8所示为1H-H COSY谱,其中化学位移值为5.35(d,J=4.15Hz,1H)的信号,说明GlcNAc与GlcA之间的键型为α键,从而证实所合成的二糖为肝素骨架。
实施例3肝素骨架合酶NaGlcNAc-T及其突变体的性质研究
(1)酶的体外反应最适反应pH测定
反应体系如表2所示,除缓冲液pH外其他都不变,将Tris-HCl缓冲液换成不同pH值的柠檬酸/磷酸盐/Tris-HCl缓冲液,分别设置4.5、5.0、5.5、6.0、6.5、7.0、7.5、8.0、8.5、9.0、9.5、10.5共计12个pH梯度点,每个梯度三组平行。其余处理条件同上。
结果如图9所示,结果说明该酶在广泛的pH范围内(6.5-9.5)具有活性,反应的最适pH为8.5。
(2)酶的体外反应最适金属离子的测定
反应体系除金属离子外其他都不变,同时将Mn2+换成相同浓度的离子Mg2+、Mn2+、Ni2+、NH4 +、Cu2+、Ca2+、K+、Ba2+、Zn2+进行反应,每种离子三个平行实验组,设置空白对照。其余处理条件同上。
结果如图10所示,结果说明,该酶在Mn2+、Mg2+、Ni2+存在的情况下均有最佳的催化活性,不加任何离子时反应程度较弱。
(3)反应温度对酶活影响的研究
反应体系如表2,设置4℃,10℃,20℃,37℃,50℃共5个温度梯度点进行酶反应,每个梯度三组平行。测定反应温度对酶活力的影响。其余处理条件同上。
结果如图11所示,表明酶反应最适的温度为37℃左右。
按照上述相同方法对六个突变体NaGlcNAc-T(C16L)、NaGlcNAc-T(N25P)、NaGlcNAc-T(I30L)、NaGlcNAc-T(I111S)、NaGlcNAc-T(S165K)、NaGlcNAc-T(S172A)的性质进行研究,发现上述六个突变体与肝素骨架合酶NaGlcNAc-T具有相似的性质,在广泛的pH范围内(6.5-9.5)具有活性,在Mn2+、Mg2+、Ni2+存在的情况下均有最佳的催化活性,不加任何离子时反应程度较弱,反应最适的温度为37℃左右。
实施例4肝素骨架合酶NaGlcNAc-T的供体特异性研究
为了确定NaGlcNAc-T的底物特异性,使用商业化GlcA-pNP作为受体,UDP-GlcNAc和其他种类结构类似的各种UDP-糖作为供体(UDP-GalNAc,UDP-Glc,UDP-Gal,UDP-GlcNTFA,UDP-GalNAz,UDP-GlcNAz、UDP-GlcNH2),按照表2体系进行反应。所有这些反应均在37℃水浴中进行4小时。最后反应产物按照表1所述的方法通过HPLC分析。
结果如图12所示,表明当受体是单糖GlcA-pNP时,在实验组所有中的单糖供体中,肝素骨架合酶NaGlcNAc-T除了其天然底物UDP-GlcNAc外,还可以以UDP-GlcNTFA、UDP-GlcNAz为底物,但反应程度较弱。
按照上述相同方法研究六个突变体NaGlcNAc-T(C16L)、NaGlcNAc-T(N25P)、NaGlcNAc-T(I30L)、NaGlcNAc-T(I111S)、NaGlcNAc-T(S165K)、NaGlcNAc-T(S172A)的供体特异性,发现上述六个突变体相对于与肝素骨架合酶NaGlcNAc-T具有相似的供体特异性,除了其天然底物UDP-GlcNAc外,还可以以UDP-GlcNTFA、UDP-GlcNAz为底物,但反应程度较弱。
SEQUENCE LISTING
<110> 山东大学
华熙生物科技股份有限公司
<120> 肝素骨架合酶及其突变体与应用
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ccgaaagaat acgcgaagta tcacaaactg aacccggtga ttccggatac cgactataag 180
gatgttggta aatttgtgca caaagttagc gataacgacg aggtgatcct gattgacgat 240
gacatcattt acccgcgtga ctatgtggaa gttctgcgtt acttctataa gaaataccaa 300
cacctgaaca tcattgttgg tacccacggc atcatttacc cggatctgta tgacggcagc 360
gtgagcagcc gtaaggtttt cacctttaaa cacagcctga agcgtccgcg tgtggttaac 420
cagctgggta ccggcaccgt gtacctgaaa ggtagccaaa tgccgagcct ggagtatatg 480
aagggcagcc agagcttcgt ggatgttcgt tttagcaaat acatgttcga aaagggtatc 540
ggcctgatct gcattccgcg tggtgcggat tggcaaaagg agatcaaaca ggaagacagc 600
attttcaaca actttaccag caaatggccg atgcaggtga tccaagaagt tcagatcatt 660
gcgggctata gcaagctgcc gtttaacctg gttaaagaga ttgaattcaa caaggacagc 720
ctgctgatga gccaa 735
<210> 6
<211> 245
<212> PRT
<213> 人工序列
<400> 6
Met Ile Ile Ala Asn Met Ala Thr Phe Pro Ala Arg Val Glu Ile Cys
1 5 10 15
Lys Gln Val Val Asp Ser Ile Tyr Pro Gln Val Asp Gln Ile Asn Leu
20 25 30
Cys Phe Asn Glu Phe Lys Gln Ile Pro Lys Glu Tyr Ala Lys Tyr His
35 40 45
Lys Leu Asn Pro Val Ile Pro Asp Thr Asp Tyr Lys Asp Val Gly Lys
50 55 60
Phe Val His Lys Val Ser Asp Asn Asp Glu Val Ile Leu Ile Asp Asp
65 70 75 80
Asp Ile Ile Tyr Pro Arg Asp Tyr Val Glu Val Leu Arg Tyr Phe Tyr
85 90 95
Lys Lys Tyr Gln His Leu Asn Ile Ile Val Gly Thr His Gly Ile Ile
100 105 110
Tyr Pro Asp Leu Tyr Asp Gly Ser Val Ser Ser Arg Lys Val Phe Thr
115 120 125
Phe Lys His Ser Leu Lys Arg Pro Arg Val Val Asn Gln Leu Gly Thr
130 135 140
Gly Thr Val Tyr Leu Lys Gly Ser Gln Met Pro Ser Leu Glu Tyr Met
145 150 155 160
Lys Gly Ser Gln Ser Phe Val Asp Val Arg Phe Ser Lys Tyr Met Phe
165 170 175
Glu Lys Gly Ile Gly Leu Ile Cys Ile Pro Arg Gly Ala Asp Trp Gln
180 185 190
Lys Glu Ile Lys Gln Glu Asp Ser Ile Phe Asn Asn Phe Thr Ser Lys
195 200 205
Trp Pro Met Gln Val Ile Gln Glu Val Gln Ile Ile Ala Gly Tyr Ser
210 215 220
Lys Leu Pro Phe Asn Leu Val Lys Glu Ile Glu Phe Asn Lys Asp Ser
225 230 235 240
Leu Leu Met Ser Gln
245
<210> 7
<211> 735
<212> DNA
<213> 人工序列
<400> 7
atgataatag ctaatatggc aacatttccc gctagggtag aaatatgtaa acaagtggtt 60
gatagcatct acaaccaggt tgaccaactg aacctgtgct tcaacgagtt taagcagatc 120
ccgaaagaat acgcgaagta tcacaaactg aacccggtga ttccggatac cgactataag 180
gatgttggta aatttgtgca caaagttagc gataacgacg aggtgatcct gattgacgat 240
gacatcattt acccgcgtga ctatgtggaa gttctgcgtt acttctataa gaaataccaa 300
cacctgaaca tcattgttgg tacccacggc atcatttacc cggatctgta tgacggcagc 360
gtgagcagcc gtaaggtttt cacctttaaa cacagcctga agcgtccgcg tgtggttaac 420
cagctgggta ccggcaccgt gtacctgaaa ggtagccaaa tgccgagcct ggagtatatg 480
aagggcagcc agagcttcgt ggatgttcgt tttagcaaat acatgttcga aaagggtatc 540
ggcctgatct gcattccgcg tggtgcggat tggcaaaagg agatcaaaca ggaagacagc 600
attttcaaca actttaccag caaatggccg atgcaggtga tccaagaagt tcagatcatt 660
gcgggctata gcaagctgcc gtttaacctg gttaaagaga ttgaattcaa caaggacagc 720
ctgctgatga gccaa 735
<210> 8
<211> 245
<212> PRT
<213> 人工序列
<400> 8
Met Ile Ile Ala Asn Met Ala Thr Phe Pro Ala Arg Val Glu Ile Cys
1 5 10 15
Lys Gln Val Val Asp Ser Ile Tyr Asn Gln Val Asp Gln Leu Asn Leu
20 25 30
Cys Phe Asn Glu Phe Lys Gln Ile Pro Lys Glu Tyr Ala Lys Tyr His
35 40 45
Lys Leu Asn Pro Val Ile Pro Asp Thr Asp Tyr Lys Asp Val Gly Lys
50 55 60
Phe Val His Lys Val Ser Asp Asn Asp Glu Val Ile Leu Ile Asp Asp
65 70 75 80
Asp Ile Ile Tyr Pro Arg Asp Tyr Val Glu Val Leu Arg Tyr Phe Tyr
85 90 95
Lys Lys Tyr Gln His Leu Asn Ile Ile Val Gly Thr His Gly Ile Ile
100 105 110
Tyr Pro Asp Leu Tyr Asp Gly Ser Val Ser Ser Arg Lys Val Phe Thr
115 120 125
Phe Lys His Ser Leu Lys Arg Pro Arg Val Val Asn Gln Leu Gly Thr
130 135 140
Gly Thr Val Tyr Leu Lys Gly Ser Gln Met Pro Ser Leu Glu Tyr Met
145 150 155 160
Lys Gly Ser Gln Ser Phe Val Asp Val Arg Phe Ser Lys Tyr Met Phe
165 170 175
Glu Lys Gly Ile Gly Leu Ile Cys Ile Pro Arg Gly Ala Asp Trp Gln
180 185 190
Lys Glu Ile Lys Gln Glu Asp Ser Ile Phe Asn Asn Phe Thr Ser Lys
195 200 205
Trp Pro Met Gln Val Ile Gln Glu Val Gln Ile Ile Ala Gly Tyr Ser
210 215 220
Lys Leu Pro Phe Asn Leu Val Lys Glu Ile Glu Phe Asn Lys Asp Ser
225 230 235 240
Leu Leu Met Ser Gln
245
<210> 9
<211> 735
<212> DNA
<213> 人工序列
<400> 9
atgataatag ctaatatggc aacatttccc gctagggtag aaatatgtaa acaagtggtt 60
gatagcatct acaaccaggt tgaccaaatt aacctgtgct tcaacgagtt taagcagatc 120
ccgaaagaat acgcgaagta tcacaaactg aacccggtga ttccggatac cgactataag 180
gatgttggta aatttgtgca caaagttagc gataacgacg aggtgatcct gattgacgat 240
gacatcattt acccgcgtga ctatgtggaa gttctgcgtt acttctataa gaaataccaa 300
cacctgaaca tcattgttgg tacccacggc tccatttacc cggatctgta tgacggcagc 360
gtgagcagcc gtaaggtttt cacctttaaa cacagcctga agcgtccgcg tgtggttaac 420
cagctgggta ccggcaccgt gtacctgaaa ggtagccaaa tgccgagcct ggagtatatg 480
aagggcagcc agagcttcgt ggatgttcgt tttagcaaat acatgttcga aaagggtatc 540
ggcctgatct gcattccgcg tggtgcggat tggcaaaagg agatcaaaca ggaagacagc 600
attttcaaca actttaccag caaatggccg atgcaggtga tccaagaagt tcagatcatt 660
gcgggctata gcaagctgcc gtttaacctg gttaaagaga ttgaattcaa caaggacagc 720
ctgctgatga gccaa 735
<210> 10
<211> 245
<212> PRT
<213> 人工序列
<400> 10
Met Ile Ile Ala Asn Met Ala Thr Phe Pro Ala Arg Val Glu Ile Cys
1 5 10 15
Lys Gln Val Val Asp Ser Ile Tyr Asn Gln Val Asp Gln Ile Asn Leu
20 25 30
Cys Phe Asn Glu Phe Lys Gln Ile Pro Lys Glu Tyr Ala Lys Tyr His
35 40 45
Lys Leu Asn Pro Val Ile Pro Asp Thr Asp Tyr Lys Asp Val Gly Lys
50 55 60
Phe Val His Lys Val Ser Asp Asn Asp Glu Val Ile Leu Ile Asp Asp
65 70 75 80
Asp Ile Ile Tyr Pro Arg Asp Tyr Val Glu Val Leu Arg Tyr Phe Tyr
85 90 95
Lys Lys Tyr Gln His Leu Asn Ile Ile Val Gly Thr His Gly Ser Ile
100 105 110
Tyr Pro Asp Leu Tyr Asp Gly Ser Val Ser Ser Arg Lys Val Phe Thr
115 120 125
Phe Lys His Ser Leu Lys Arg Pro Arg Val Val Asn Gln Leu Gly Thr
130 135 140
Gly Thr Val Tyr Leu Lys Gly Ser Gln Met Pro Ser Leu Glu Tyr Met
145 150 155 160
Lys Gly Ser Gln Ser Phe Val Asp Val Arg Phe Ser Lys Tyr Met Phe
165 170 175
Glu Lys Gly Ile Gly Leu Ile Cys Ile Pro Arg Gly Ala Asp Trp Gln
180 185 190
Lys Glu Ile Lys Gln Glu Asp Ser Ile Phe Asn Asn Phe Thr Ser Lys
195 200 205
Trp Pro Met Gln Val Ile Gln Glu Val Gln Ile Ile Ala Gly Tyr Ser
210 215 220
Lys Leu Pro Phe Asn Leu Val Lys Glu Ile Glu Phe Asn Lys Asp Ser
225 230 235 240
Leu Leu Met Ser Gln
245
<210> 11
<211> 735
<212> DNA
<213> 人工序列
<400> 11
atgataatag ctaatatggc aacatttccc gctagggtag aaatatgtaa acaagtggtt 60
gatagcatct acaaccaggt tgaccaaatt aacctgtgct tcaacgagtt taagcagatc 120
ccgaaagaat acgcgaagta tcacaaactg aacccggtga ttccggatac cgactataag 180
gatgttggta aatttgtgca caaagttagc gataacgacg aggtgatcct gattgacgat 240
gacatcattt acccgcgtga ctatgtggaa gttctgcgtt acttctataa gaaataccaa 300
cacctgaaca tcattgttgg tacccacggc atcatttacc cggatctgta tgacggcagc 360
gtgagcagcc gtaaggtttt cacctttaaa cacagcctga agcgtccgcg tgtggttaac 420
cagctgggta ccggcaccgt gtacctgaaa ggtagccaaa tgccgagcct ggagtatatg 480
aagggcagcc agaaattcgt ggatgttcgt tttagcaaat acatgttcga aaagggtatc 540
ggcctgatct gcattccgcg tggtgcggat tggcaaaagg agatcaaaca ggaagacagc 600
attttcaaca actttaccag caaatggccg atgcaggtga tccaagaagt tcagatcatt 660
gcgggctata gcaagctgcc gtttaacctg gttaaagaga ttgaattcaa caaggacagc 720
ctgctgatga gccaa 735
<210> 12
<211> 245
<212> PRT
<213> 人工序列
<400> 12
Met Ile Ile Ala Asn Met Ala Thr Phe Pro Ala Arg Val Glu Ile Cys
1 5 10 15
Lys Gln Val Val Asp Ser Ile Tyr Asn Gln Val Asp Gln Ile Asn Leu
20 25 30
Cys Phe Asn Glu Phe Lys Gln Ile Pro Lys Glu Tyr Ala Lys Tyr His
35 40 45
Lys Leu Asn Pro Val Ile Pro Asp Thr Asp Tyr Lys Asp Val Gly Lys
50 55 60
Phe Val His Lys Val Ser Asp Asn Asp Glu Val Ile Leu Ile Asp Asp
65 70 75 80
Asp Ile Ile Tyr Pro Arg Asp Tyr Val Glu Val Leu Arg Tyr Phe Tyr
85 90 95
Lys Lys Tyr Gln His Leu Asn Ile Ile Val Gly Thr His Gly Ile Ile
100 105 110
Tyr Pro Asp Leu Tyr Asp Gly Ser Val Ser Ser Arg Lys Val Phe Thr
115 120 125
Phe Lys His Ser Leu Lys Arg Pro Arg Val Val Asn Gln Leu Gly Thr
130 135 140
Gly Thr Val Tyr Leu Lys Gly Ser Gln Met Pro Ser Leu Glu Tyr Met
145 150 155 160
Lys Gly Ser Gln Lys Phe Val Asp Val Arg Phe Ser Lys Tyr Met Phe
165 170 175
Glu Lys Gly Ile Gly Leu Ile Cys Ile Pro Arg Gly Ala Asp Trp Gln
180 185 190
Lys Glu Ile Lys Gln Glu Asp Ser Ile Phe Asn Asn Phe Thr Ser Lys
195 200 205
Trp Pro Met Gln Val Ile Gln Glu Val Gln Ile Ile Ala Gly Tyr Ser
210 215 220
Lys Leu Pro Phe Asn Leu Val Lys Glu Ile Glu Phe Asn Lys Asp Ser
225 230 235 240
Leu Leu Met Ser Gln
245
<210> 13
<211> 735
<212> DNA
<213> 人工序列
<400> 13
atgataatag ctaatatggc aacatttccc gctagggtag aaatatgtaa acaagtggtt 60
gatagcatct acaaccaggt tgaccaaatt aacctgtgct tcaacgagtt taagcagatc 120
ccgaaagaat acgcgaagta tcacaaactg aacccggtga ttccggatac cgactataag 180
gatgttggta aatttgtgca caaagttagc gataacgacg aggtgatcct gattgacgat 240
gacatcattt acccgcgtga ctatgtggaa gttctgcgtt acttctataa gaaataccaa 300
cacctgaaca tcattgttgg tacccacggc atcatttacc cggatctgta tgacggcagc 360
gtgagcagcc gtaaggtttt cacctttaaa cacagcctga agcgtccgcg tgtggttaac 420
cagctgggta ccggcaccgt gtacctgaaa ggtagccaaa tgccgagcct ggagtatatg 480
aagggcagcc agagcttcgt ggatgttcgt tttgcaaaat acatgttcga aaagggtatc 540
ggcctgatct gcattccgcg tggtgcggat tggcaaaagg agatcaaaca ggaagacagc 600
attttcaaca actttaccag caaatggccg atgcaggtga tccaagaagt tcagatcatt 660
gcgggctata gcaagctgcc gtttaacctg gttaaagaga ttgaattcaa caaggacagc 720
ctgctgatga gccaa 735
<210> 14
<211> 245
<212> PRT
<213> 人工序列
<400> 14
Met Ile Ile Ala Asn Met Ala Thr Phe Pro Ala Arg Val Glu Ile Cys
1 5 10 15
Lys Gln Val Val Asp Ser Ile Tyr Asn Gln Val Asp Gln Ile Asn Leu
20 25 30
Cys Phe Asn Glu Phe Lys Gln Ile Pro Lys Glu Tyr Ala Lys Tyr His
35 40 45
Lys Leu Asn Pro Val Ile Pro Asp Thr Asp Tyr Lys Asp Val Gly Lys
50 55 60
Phe Val His Lys Val Ser Asp Asn Asp Glu Val Ile Leu Ile Asp Asp
65 70 75 80
Asp Ile Ile Tyr Pro Arg Asp Tyr Val Glu Val Leu Arg Tyr Phe Tyr
85 90 95
Lys Lys Tyr Gln His Leu Asn Ile Ile Val Gly Thr His Gly Ile Ile
100 105 110
Tyr Pro Asp Leu Tyr Asp Gly Ser Val Ser Ser Arg Lys Val Phe Thr
115 120 125
Phe Lys His Ser Leu Lys Arg Pro Arg Val Val Asn Gln Leu Gly Thr
130 135 140
Gly Thr Val Tyr Leu Lys Gly Ser Gln Met Pro Ser Leu Glu Tyr Met
145 150 155 160
Lys Gly Ser Gln Ser Phe Val Asp Val Arg Phe Ala Lys Tyr Met Phe
165 170 175
Glu Lys Gly Ile Gly Leu Ile Cys Ile Pro Arg Gly Ala Asp Trp Gln
180 185 190
Lys Glu Ile Lys Gln Glu Asp Ser Ile Phe Asn Asn Phe Thr Ser Lys
195 200 205
Trp Pro Met Gln Val Ile Gln Glu Val Gln Ile Ile Ala Gly Tyr Ser
210 215 220
Lys Leu Pro Phe Asn Leu Val Lys Glu Ile Glu Phe Asn Lys Asp Ser
225 230 235 240
Leu Leu Met Ser Gln
245
Claims (8)
1.一种肝素骨架合酶突变体NaGlcNAc-T(C16L),其特征在于,其氨基酸序列如SEQ IDNO.4所示,编码基因的核苷酸序列如SEQ ID NO.3所示;是将SEQ ID NO.2所示的肝素骨架合酶氨基酸序列中第16位的半胱氨酸定点突变为亮氨酸。
2.一种肝素骨架合酶突变体NaGlcNAc-T(S165K),其特征在于,其氨基酸序列如SEQ IDNO.12所示,编码基因的核苷酸序列如SEQ ID NO.11所示;是将SEQ ID NO.2所示的肝素骨架合酶氨基酸序列中第165位的丝氨酸定点突变为赖氨酸。
3.一种重组载体,其特征在于,是在质粒载体中插入权利要求1或权利要求2所述的肝素骨架合酶突变体的编码基因。
4.如权利要求3所述的重组载体,其特征在于,所述质粒载体为pET30a(+)。
5.一种重组菌株,其特征在于,是将权利要求3所述的重组载体转化入宿主细胞中获得。
6.如权利要求5所述的重组菌株,其特征在于,所述宿主细胞为大肠杆菌。
7.权利要求1或权利要求2所述的肝素骨架合酶突变体在合成肝素糖链中的应用。
8.如权利要求7所述的应用,其特征在于,所述应用是以GlcA-pNP作为起始受体,UDP-GlcNAc作为供体,生成结构为GlcNAc-GlcA-pNP的肝素骨架二糖。
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CN104017765A (zh) * | 2014-06-16 | 2014-09-03 | 山东大学 | 一株表面具有Galα1-4Galβ1-4GlcNAc结构的工程菌及其应用 |
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