Biology
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Summary May 14th, 2021
Please note that all translations are automatically generated.
Click here for the English version.
本研究为分析E3泛素连接酶催化活性提供了详细的 体外 泛素化测定方案。重组蛋白使用原核系统（如 大肠 杆菌培养物）表达。
Transcript
该协议的总体目标是分析E3连接酶功能的重要关键方面，包括E2酶特异性，底物选择和泛素转移。该技术的主要优点是其广泛的适用性，因为来自所有真核来源的蛋白质可以在体外重组表达和研究，而无需特殊设备。首次使用赖氨酸放电技术时，重要的是优化测定参数，例如酶浓度，以确定所选E3连接酶的理想放电条件。
为了进行体外自动泛素化测定，建立移液方案以测试不同E2酶的功能能力，并在冰上制备用于所有泛素化反应和一个额外反应的预混液。接下来，将一微摩尔E2酶加入适当的试管中，并在指定的条件下将样品在PCR热循环仪中孵育两小时。孵育后，向每个反应中加入SDS样品缓冲液，并通过移液多次混合。
然后立即将样品在95摄氏度下煮沸五分钟，并将变性蛋白质储存在20摄氏度。为了进行体外底物泛素化测定，请为所有反应准备移液方案。在计算一次反应所需的预混液量后，在冰上制备所有反应的预混液，并将预混液加入每个管中。
单独添加相应的 E3 连接酶。然后如图所示，将样品在PCR热循环仪中孵育两小时。在孵育结束时，向每个反应中加入两次SDS样品缓冲液，通过移液混合几次，并在95摄氏度下将样品煮沸五分钟。
要进行赖氨酸放电测定，请设置装药反应的移液方案，并将装药反应在37摄氏度下孵育15分钟。为了停止充电反应，将烧焦酶加入到每毫升1.8单位的终浓度中，并在室温下孵育反应五分钟。在孵育结束时，将EDTA加入至终浓度为30毫摩尔，并使用双蒸馏水将反应体积调节至30微升。
为了通过E3放电E2泛素化，设置五个对应于实验时间点的管，向每个管中加入6.7微升非还原性样品缓冲液，从时间点零管中除去6微升停止充电反应，并将总体积调节至26.7微升。为了建立放电反应，向每个反应中加入双蒸馏水，泛素化缓冲液，BSA，赖氨酸，E3连接酶和带电的E2。在选定的时间段后，将20微升样品从每次放电反应转移到相应的样品管中。
然后立即涡旋样品，并将它们置于70摄氏度下10分钟。在这项具有代表性的蛋白质印迹分析中，非活性芯片不是自动泛素化的。然而，E3非依赖性的泛素产物是在存在非活性CHIP和没有CHIP的情况下形成的。
野生型CHIP与UBE2D和UBE2E家族的蛋白质结合时是自泛素化的，而游离的多泛素链是与UBE2D家族的蛋白质合作生产的，而不是与UBE2E家族的蛋白质合作生产的。UBE2N/V1对泛素的结合引导游离泛素链的形成。使用野生型CHIP观察到UNC-45B的自泛素化和泛素化，但未观察到CHIP的非活性突变体，这表明UNC-45B充当CHIP的保守底物。
当使用赖氨酸放电测定法分析CHIP的催化活性时，未带电的UBE2D2酶的分子量为17千道尔顿，具有单个泛素分子的带电UBE2D2的分子量约为26千道尔顿。在时间 0 处，观察到整个带电的 E2 当量。在存在非活性CHIP的情况下，检测到UBE2D2的微弱E3连接酶非依赖性放电，但未检测到CHIP的自泛素化。
在野生型CHIP存在下，UBE2D2的放电速度快，在60分钟内完成，CHIP的自泛素化表明泛素转移到其自身赖氨酸残基上。由于E2能力有限，请尽快工作并立即进行放电反应，然后进行SDS-PAGE和蛋白质印迹。遵循这些体外泛素化方案，可以通过用连锁特异性抗体探测蛋白质印迹来鉴定特定的泛素化连锁类型。
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