A multidomain PARP14 construct suitable for bacterial expression

IF 1.4 4区生物学 Q4 BIOCHEMICAL RESEARCH METHODS Protein expression and purification Pub Date : 2024-08-17 DOI:10.1016/j.pep.2024.106580

Constantinos Chatzicharalampous, Herwig Schüler

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Abstract

Poly-ADP-ribose polymerase-14 (PARP14) can modify proteins and nucleic acids by the reversible addition of a single ADP-ribose molecule. Aberrant PARP14 functions have been related to cancer and inflammation, and its domains are involved in processes related to viral infection. Previous research indicates that PARP14 functions might be mediated via a multitude of target proteins. In vitro studies of this large multidomain enzyme have been complicated by difficulties to obtain biochemical quantities of pure protein. Here we present a strategy that allows bacterial expression and purification of a functional multidomain construct of PARP14. We substituted an internal KH domain and its neighboring unstructured region with a SUMO domain to obtain a protein construct that encompasses three macrodomains, a WWE domain, and a PARP catalytic domain. We show that the resulting construct retains both ADP-ribosyltransferase and de-MARylase activities. This construct will be useful in structural and functional studies of PARP14.

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适合细菌表达的多域 PARP14 构建物。

聚 ADP 核糖聚合酶-14（Poly-ADP-Rribose polymerase-14，PARP14）可以通过可逆添加单个 ADP 核糖分子来修饰蛋白质和核酸。PARP14 的功能异常与癌症和炎症有关，其结构域也参与了与病毒感染有关的过程。以前的研究表明，PARP14 的功能可能是通过多种靶蛋白介导的。由于难以获得生化数量的纯蛋白，对这种大型多结构域酶的体外研究一直很复杂。在这里，我们提出了一种策略，可以通过细菌表达和纯化 PARP14 的功能性多域构建体。我们用一个 SUMO 结构域取代了一个内部 KH 结构域及其邻近的非结构化区域，从而获得了一个包含三个大结构域、一个 WWE 结构域和一个 PARP 催化结构域的蛋白质构建体。我们的研究表明，这种构建体保留了 ADP 核糖基转移酶和去甲基化酶的活性。该构建体将有助于 PARP14 的结构和功能研究。

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来源期刊

Protein expression and purification 生物-生化研究方法

CiteScore

3.70

自引率

6.20%

发文量

120

审稿时长

32 days

期刊介绍： Protein Expression and Purification is an international journal providing a forum for the dissemination of new information on protein expression, extraction, purification, characterization, and/or applications using conventional biochemical and/or modern molecular biological approaches and methods, which are of broad interest to the field. The journal does not typically publish repetitive examples of protein expression and purification involving standard, well-established, methods. However, exceptions might include studies on important and/or difficult to express and/or purify proteins and/or studies that include extensive protein characterization, which provide new, previously unpublished information.