Department of Cellular and Molecular Biophysics, Max Planck Institute of Biochemistry, D-82152 Martinsried, Germany.
School of Life Sciences, Jiangsu Normal University, Shanghai Road 101, 221116 Xuzhou, P. R. China.
ACS Synth Biol. 2023 Jan 20;12(1):108-119. doi: 10.1021/acssynbio.2c00406. Epub 2022 Nov 29.
Reversible membrane targeting of proteins is one of the key regulators of cellular interaction networks, for example, for signaling and polarization. So-called "membrane switches" are thus highly attractive targets for the design of minimal cells but have so far been tricky to reconstitute in vitro. Here, we introduce cell-free prenylated protein synthesis (CFpPS), which enables the synthesis and membrane targeting of proteins in a single reaction mix including the prenylation machinery. CFpPS can confer membrane affinity to any protein via addition of a 4-peptide motif to its C-terminus and offers robust production of prenylated proteins not only in their soluble forms but also in the direct vicinity of biomimetic membranes. Thus, CFpPS enabled us to reconstitute the prenylated polarity hub Cdc42 and its regulatory protein in vitro, implementing a key membrane switch. We propose CFpPS to be a versatile and effective platform for engineering complex features, such as polarity induction, in synthetic cells.
蛋白质的可还原膜靶向是细胞相互作用网络的关键调节剂之一，例如用于信号转导和极化。因此，所谓的“膜开关”是设计最小细胞的极具吸引力的目标，但迄今为止在体外很难重新构成。在这里，我们引入了无细胞香叶酰化蛋白合成 (CFpPS)，它可以在包括香叶酰化机制在内的单个反应混合物中合成和靶向蛋白质。CFpPS 可以通过在其 C 末端添加 4 肽基序来赋予任何蛋白质膜亲和力，并提供稳定的香叶酰化蛋白生产，不仅可以以可溶性形式，还可以在生物模拟膜的直接附近进行。因此，CFpPS 使我们能够在体外重新构建香叶酰化极性枢纽 Cdc42 及其调节蛋白，实现了关键的膜开关。我们提出 CFpPS 是一种通用且有效的平台，可用于工程合成细胞中的复杂特征，例如极性诱导。
