CN116693610A - 用于测定多肽连接酶的探针分子、测定转肽酶a的方法及应用 - Google Patents
用于测定多肽连接酶的探针分子、测定转肽酶a的方法及应用 Download PDFInfo
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Abstract
本发明提供了一种用于测定多肽连接酶的探针分子,及测定转肽酶A的方法及应用。该AIE多肽探针结构为DDLPETGGFK‑TPE,可被SrtA催化发生转肽反应,释放出来的含有AIE基团的片段将会迅速聚集并发出荧光。该检测方法能够降低连接酶催化反应的可逆性,提高底物的转化效率,同时实现连接酶的灵敏检测和实时成像,从而解决了传统连接酶荧光探针存在的信噪比低、底物结构复杂、转化效率低、灵敏度差等问题。
Description
技术领域
本发明具体涉及一种用于测定多肽连接酶的探针分子、测定转肽酶A的方法及应用,属于生物化学检测技术领域。
背景技术
多肽连接酶在蛋白质工程、化学标记和蛋白质环化等方面发挥着重要作用,包括转肽酶(sortase)、丁酰胺连接酶-1、天冬酰胺连接酶OaAEP1、VyPAL2、枯草杆菌酶变体等等,它们的活性检测及其抑制剂筛选在生物学研究、医学诊断和药物开发等领域具有重要意义。目前用于多肽连接酶活性检测的方法主要包括高效液相色谱法、质谱法和荧光分析法等。相对而言,荧光分析法具有灵敏度高、操作简单等优点,包括荧光共振能量转移(FRET)、酵母表面展示(YSD)荧光分析、蛋白质片段互补测定等。但这些方法存在信噪比低、底物结构复杂等问题。此外,由于多肽连接酶催化的反应是完全可逆的(酶促反应的产物同时也是多肽连接酶的底物),因此,传统的荧光探针在检测多肽连接酶时还存在底物转化率低、灵敏度差等问题。通过增加副反应物的浓度和去除副产物的方式(包括透析、萃取、副产物灭活等)可以有效降低连接酶催化反应的可逆性,提高底物的转化效率。然而,这些方法均不适合用于设计检测连接酶的荧光探针。因此,为了实现多肽连接酶的灵敏检测和实时成像,仍然迫切需要开发具有更高催化效率的新策略。介于多肽连接酶以其独特的催化肽键形成的能力,促进了蛋白质修饰的发展,使其具有优良的位点特异性、高的反应效率和非凡的通用性,建立一种低成本、简单快速、适于在一般实验室或临床中监控多肽连接酶活性变化的方法是当前研究的热点之一。
聚集诱导发光(AIE)是指物质在溶液中不发光或发光微弱,而在聚集或固态状态下显著发光的现象。相对于传统的荧光材料,AIE材料具有稳定性好、背景信号低、抗光漂白能力强等优点,已在光电材料、生物医学、荧光传感等领域展现出广泛的应用前景。其中,具有AIE效应的荧光探针已成功用于蛋白酶、磷酸化酶、糖苷酶、胆碱酶、端粒酶等生物酶的检测。针对多肽连接酶催化反应的可逆性问题,大多数报道的连接酶检测的分析方法不能通过降低连接反应的可逆性来提高催化效率。因此,开发一种灵敏经济高效的AIE荧光方法来检测多肽连接酶活性、提高转化效率、降低酶反应的可逆性是十分必要的。
发明内容
本发明的目的在于克服目前的多肽连接酶检测中存在的上述问题,提供一种用于测定多肽连接酶的探针分子、测定转肽酶A的方法及应用。
本发明的技术方案如下:
一种用于测定多肽连接酶的探针分子,该探针分子具有水溶性好且在溶液中不发光的特点,其包括多肽部分和AIE部分;所述多肽部分可被多肽连接酶识别切割发生酶催化反应;所述AIE部分可对多肽部分进行修饰,且在多肽被多肽连接酶催化发生转肽反应后,可释放出含有AIE基团的疏水片段且该片段会迅速聚集并产生荧光。
进一步的,所述多肽连接酶为转肽酶A,所述多肽部分包括LPXTG序列,其中X表示任意氨基酸残基;所述AIE部分为四苯基乙烯(TPE)及其衍生物。
进一步的,所述探针分子的序列结构为DDLPETGGFK-TPE。
一种测定转肽酶A的方法,将探针分子与底物肽GGG按一定比例混合得到混合溶液,向该混合溶液中加入待侧的转肽酶A溶液,混合孵育,得到反应产物GGFK-TPE,利用荧光光谱仪检测其在460nm处的荧光强度,根据标准曲线得到待侧转肽酶A溶液的活性浓度。
另外,本发明还提供了上述测定转肽酶A的方法在测定革兰氏阳性菌中SrtA的检测应用,以及在筛选潜在的靶向SrtA抗病毒抑制剂药物中的应用。
有益效果:本发明提供一种用于测定多肽连接酶的探针分子,及一种对多肽连接酶活性高效检测的AIE荧光分析方法。该方法选择了几乎所有革兰氏阳性菌中都存在的SrtA为模型分析物,设计合成AIE多肽探针。该多肽探针分子的水溶性较好,在溶液中不发光;一旦多肽被SrtA催化发生转肽反应之后,释放出来的含有AIE基团的片段将会迅速聚集,AIE分子聚集组装之后,溶液中AIE单体(酶催化产物)的浓度将会大大降低,从而使得酶催化反应不可逆,提高底物的转化效率,实现SrtA的聚集诱导发光检测和实时成像的目的。该检测方法能够降低连接酶催化反应的可逆性,提高底物的转化效率,同时实现连接酶的灵敏检测,从而解决了传统连接酶荧光探针存在的信噪比低、底物结构复杂、转化效率低、灵敏度差等问题。
附图说明
图1是SrtA检测的实验原理图。
图2是SrtA检测可行性的荧光发射光谱图。
图3 GGFK-TPE聚集体的粒度分析和透射电镜表征图。
图4 采用高效液相色谱分析SrtA活性的色谱图。
图5是不同浓度SrtA存在下产物的荧光发射光谱检测。
图6是抑制剂对SrtA抑制效率的测定。
图7是对其它蛋白或酶检测的选择性。
图8是测定复杂生物样品中SrtA的结果。
图9是测定金黄色葡萄球菌中SrtA的结果。
实施方式
为了更充分的解释本发明的实施,提供本发明的实施实例。这些实施实例仅仅是对该工艺的阐述,不限制本发明的范围,本发明中用以下实施例说明,但不限于下述实施例,任何根据本发明的核心思想而推得的变化实施都包含在本发明的技术范围内。
本发明的主要构思如下:采用四苯基乙烯(TPE)衍生物作为骨架用于构筑AIE探针,并对多肽进行修饰。所用多肽的序列特征是:具有能够被SrtA识别并切割的酶催化反应的C端LPXTG序列的多肽并与其发生连接反应(其中X表示任意氨基酸残基),用于SrtA的灵敏检测。
用于SrtA灵敏检测的AIE方法,采用上述的多肽功能化AIE探针分子,包括以下步骤:
A:多肽探针的分子设计:TPE衍生物结构简单、易于合成、聚集发光效果较好,是一种经典的AIE分子,常被作为骨架用于构筑AIE探针。因此,采用TPE衍生物对多肽进行修饰。如图1所示,基于AIE探针聚集的特征及SrtA转肽酶催化反应的特点,设计的多肽探针序列为DDLPETGGFK-TPE。该多肽的LPETG部分能够被SrtA识别,是酶催化反应的活性位点;N-端的两个天冬氨酸残基(DD)可以增强多肽底物的水溶性,避免多肽底物的聚集;TPE基团修饰在C端赖氨酸残基(K)的侧链,并在C端引入苯丙氨酸残基(F),以增强酶催化产物(GGFK-TPE)的疏水性,从而有利于催化产物的聚集组装。实验过程中,多肽探针经过与甘氨酸亲核试剂的GGG片段反应之后,释放出GGFK-TPE片段,该产物聚集自组装发出荧光。该聚集体的形成降低了溶液中GGFK-TPE单体的浓度和竞争基团的亲和性,使酶促反应平衡向生成物方向移动,降低了可逆性,提高了催化效率。
B:SrtA的定量检测:采用荧光光谱、高效液相色谱研究SrtA催化多肽探针与底物肽GGG发生转肽反应的可行性;研究该方法的线性范围、灵敏度、选择性、抗干扰能力等分析性能;测定葡萄球菌细胞中SrtA的活性,考察抑制剂对SrtA活性的抑制效率。
本发明中多肽序列为DDLPETGGFK-TPE,该多肽的LPETG部分能够被SrtA识别,是酶催化反应的活性位点。以下实施例以SrtA为例进行具体阐述。对附图进一步的解释,图1是转肽酶A(SrtA)检测的实验原理图。图2是DDLPETGGFK-TPE(曲线a)、GGFK-TPE(曲线b)、DDLPETGGFK-TPE + SrtA(曲线c)和DDLPETGGFK-TPE + SrtA + GGG(曲线d)在460 nm处的荧光发射光谱图。图3是用粒度分析仪(DLS)和透射电镜(TEM)表征GGFK-TPE聚集体;图4是采用高效液相色谱分析DDLPETGGFK-TPE、DDLPETGGFK-TPE + SrtA + GGGSrtA和GGFK-TPE溶液的色谱图。图5(A)是SrtA的浓度为0~200 nM时于360~600 nm范围内的荧光发射光谱图;(B)是SrtA的浓度为0~200 nM时在460 nm处的荧光强度图,其中的插图为SrtA在0~50nM范围内和荧光强度的线性关系图。图6是SrtA的抑制剂氯化小檗碱和槲皮素(berberinechloride)和槲皮素(quercetin)对荧光强度的影响情况。图7是选择性实验,与SrtA共存的蛋白质和酶分别为牛血清白蛋白(BSA), 碱性磷酸酶(ALP),溶菌酶(lysozyme),胰蛋白酶(trypsin)和乳糜酶(chymase);图8是在复杂生物样品中对SrtA的检测结果,SrtA 的浓度分别为0.1, 1 和 10 nM,检测体系分别为缓冲溶液,血清和细胞裂解液。图9是测定有无抑制剂氯化小檗碱时的金黄色葡萄球菌中SrtA的结果。
实施例1:可行性研究
分别将DDLPETGGFK-TPE 和 GGFK-TPE溶解在DMSO中制成5 mM浓度,采用缓冲液稀释后在312 nm下激发,记录360 至 600 nm范围内的荧光光谱。从图2中可以看出,DDLPETGGFK-TPE几乎无荧光,GGFK-TPE在460 nm处发出较强荧光。DDLPETGGFK-TPE + SrtA几乎无荧光,DDLPETGGFK-TPE + SrtA+GGG发出较强荧光,且荧光强度在90分钟时趋于平稳。实验结果表明,通过监测GGFK-TPE聚集体的形成及荧光发射强度可用于SrtA的测定,操作步骤简单,连接反应效率高。用DLS和TEM表征了GGFK-TPE的尺寸和分布情况,实验结果如图3所示。
实施例2:HPLC分析
采用高效液相色谱分析了SrtA的活性,底物和酶促反应的产物用C-18柱分离,流动相为50%的CH3OH,流速为1 mL/分钟,流动相在使用之前用0.22 μm的滤膜过滤。记录信号的波长为254 nm。用于连接反应的SrtA为1 μM,加入底物DDLPETGGFK-TPE (25 μM) 和 GGG(50 μM),于反应缓冲液中,25°C下反应2 h,之后于95 °C 下加热20 分钟以终止反应。在进行高效液相色谱分离之前,采用等体积的CH3OH稀释溶液,将聚集体进行溶解。从图4中可以看出,DDLPETGGFK-TPE几乎全部发生催化转化反应,产生GGFK-TPE。
实施例3:SrtA的检测
向含有10 μM DDLPETGGFK-TPE和20 μM GGG的90 µL反应缓冲液中分别加入10 µL不同浓度的SrtA,于25 °C下反应120 分钟,之后于312 nm激发波长下记录其荧光发射光谱,结果如图5所示。随着SrtA浓度的增加,460 nm 处的荧光强度也相应增加,且在0~50 nM范围内呈现良好的线性关系,检出限低至12 pM。这归因于TPE良好的自组装能力和聚集发光性能,以及该反应体系较高的连接效率。向反应溶液中加入其它蛋白质或酶代替SrtA,其它测试步骤与SrtA标准样品的测定相同,考察了实验方案对SrtA的选择性,结果如图7所示。在其它干扰物存在的情况下,该实验方案对SrtA仍然有较高的选择性。如图8所示,即使在存在多种干扰物质的生物基质样品(如血清、乳腺癌细胞裂解液)中,本实验方案仍具有检测SrtA的高度特异性。
实施例4:抑制分析
分别用不同浓度的抑制剂氯化小檗碱或槲皮素和50 nM的SrtA孵育5分钟,之后将10 µL的上述混合液加入到90 µL含有底物的反应缓冲液中,并于25 ◦C下孵育2小时。记录了其在460 nm下荧光强度的下降情况。结果如图6所示,其IC50分别为47.8 μM和8.3 μM。该抑制率实验表明,本实验方案可用于筛选潜在的抑制剂药物。
实施例5:金黄色葡萄球菌中SrtA的检测
金黄色葡萄球菌用BHI培养基于37 °C 和 200 rpm的摇床中培养,采用平板计数法计算金黄色葡萄球菌的浓度,用细菌蛋白抽提试剂提取细胞中的SrtA。超声处理破坏细胞之后,10000 rpm离心20分钟,收集上清液,用反应缓冲液稀释。取10 µL稀释后的上清液孵育90 µL的DDLPETGGFK-TPE/GGG 混合物,并于 25 °C下反应2小时,之后在荧光光谱仪上记录其荧光发射光谱。将抑制剂氯化小檗碱加入到培养基中,使其最终浓度为25 μM,检测细菌悬浮液在600 nm处的光密度,发现氯化小檗碱对金黄色葡萄球菌的生长没有明显的抑制作用。抑制剂处理的金黄色葡萄球菌细胞中SrtA的提取和检测方法与未添加氯化小檗碱的方法相同。结果如图9所示。实验表明,该抑制剂可作为一种潜在的靶向SrtA的抗病毒药物,且本实验方案可用于革兰氏阳性细菌中SrtA的活性监测。
本发明的机理探讨:本实验以转肽酶SrtA为模型分析物,设计合成的AIE多肽探针DDLPETGGFK-TPE水溶性较好,在溶液中几乎无荧光。被SrtA催化发生转肽反应之后,释放出来的含有AIE基团的片段GGFK-TPE水溶性较差,会迅速聚集。该聚集体的形成,可以提高连接反应的效率和灵敏度,从而使得酶催化反应不可逆,实现SrtA的聚集诱导发光检测。该探针在其它蛋白质和酶存在下对SrtA仍具有良好的选择性,可用于生物样品和金黄色葡萄球菌细胞中的SrtA检测,有助于建立用于SrtA抑制剂药物筛选的方法。
在详细说明本发明的实施方式之后,熟悉该项技术的人士可清楚地了解,在不脱离上述申请专利范围与精神下可进行各种变化与修改,凡依据本发明的技术实质对以上实施例所作的任何简单修改、等同变化与修饰,均属于本发明技术方案的范围,且本发明亦不受限于说明书中所举实例的实施方式。
Claims (5)
1.一种用于测定多肽连接酶的探针分子,其特征在于:所述探针分子具有水溶性好且在溶液中不发光的特点,其包括多肽部分和AIE部分;所述多肽部分可被多肽连接酶识别切割发生酶催化反应;所述AIE部分可对多肽部分进行修饰,且在多肽被多肽连接酶催化发生转肽反应后,可释放出含有AIE基团的疏水片段且该片段会迅速聚集并产生荧光。
2.根据权利要求1所述的一种用于测定多肽连接酶的探针分子,其特征在于:所述多肽连接酶为转肽酶A,所述多肽部分包括LPXTG序列,其中X表示任意氨基酸残基;所述AIE部分为四苯基乙烯(TPE)及其衍生物。
3.根据权利要求2所述的一种用于测定多肽连接酶的探针分子,其特征在于:所述探针分子的序列结构为DDLPETGGFK-TPE。
4.一种测定转肽酶A的方法,将权利要求3所述的探针分子与底物肽GGG按一定比例混合得到混合溶液,向该混合溶液中加入待侧的转肽酶A溶液,混合孵育,得到反应产物GGFK-TPE,利用荧光光谱仪检测其在460nm处的荧光强度,根据标准曲线得到待侧转肽酶A溶液的活性浓度。
5.如权利要求4所述的测定转肽酶A的方法在测定革兰氏阳性菌中SrtA的检测中的应用,或在筛选潜在的靶向SrtA抗病毒抑制剂药物中的应用。
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