Abstract：
Expression and purification of membrane-bound proteins remains a challenge and limits enzymology efforts, contributing to a substantial knowledge gap in the biochemical functions of many proteins found in nature. Accordingly, the study of bacterial UbiA terpene synthases (TSs) has been limited due to the experimental hurdles required to purify active enzymes for characterization in vitro. Previous work employed the use of microsomes or crude membrane fractions to test enzyme activity; however, these methods can be labor intensive, require access to an ultracentrifuge, or may not be suitable for all membrane-bound TSs. We detail here an alternative strategy for the in vivo expression and biochemical characterization of the membrane associated UbiA TSs by employing a precursor overproduction system in Escherichia coli.
摘要：
膜结合蛋白的表达和纯化仍然是一个挑战，限制了酶学的努力。在自然界中发现的许多蛋白质的生化功能方面造成了巨大的知识空白。因此，由于纯化体外表征活性酶所需的实验障碍,细菌UbiA萜烯合酶(TS)的研究受到限制。以前的工作采用微粒体或粗膜部分来测试酶活性；然而，这些方法可能是劳动密集型的，需要使用超速离心机，或者可能不适用于所有膜结合TS。我们在这里详细介绍了通过在大肠杆菌中采用前体过量生产系统来实现膜相关UbiATS的体内表达和生化表征的替代策略。
