Reversible phosphorylation is a fundamental mechanism for controlling protein function. Despite the critical roles phosphorylated proteins play in physiology and disease, our ability to study individual phospho-proteoforms has been hindered by a lack of versatile methods to efficiently generate homogeneous proteins with site-specific phosphoamino acids or with functional mimics that are resistant to phosphatases. Genetic code expansion (GCE) is emerging as a transformative approach to tackle this challenge, allowing direct incorporation of phosphoamino acids into proteins during translation in response to amber stop codons. This genetic programming of phospho-protein synthesis eliminates the reliance on kinase-based or chemical semisynthesis approaches, making it broadly applicable to diverse phospho-proteoforms. In this comprehensive review, we provide a brief introduction to GCE and trace the development of existing GCE technologies for installing phosphoserine, phosphothreonine, phosphotyrosine, and their mimics, discussing both their advantages as well as their limitations. While some of the technologies are still early in their development, others are already robust enough to greatly expand the range of biologically relevant questions that can be addressed. We highlight new discoveries enabled by these GCE approaches, provide practical considerations for the application of technologies by non-GCE experts, and also identify avenues ripe for further development.

中文翻译：

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磷酸化氨基酸基因编码为蛋白质

可逆磷酸化是控制蛋白质功能的基本机制。尽管磷酸化蛋白质在生理学和疾病中发挥着关键作用，但由于缺乏有效生成具有位点特异性磷酸氨基酸或具有抗磷酸酶的功能模拟物的均质蛋白质的通用方法，我们研究个体磷酸化蛋白质形式的能力受到阻碍。遗传密码扩展（GCE）正在成为应对这一挑战的变革性方法，允许在翻译过程中响应琥珀终止密码子将磷酸氨基酸直接掺入蛋白质中。这种磷酸蛋白合成的遗传编程消除了对基于激酶或化学半合成方法的依赖，使其广泛适用于各种磷酸蛋白形式。在这篇综合综述中，我们简要介绍了 GCE，并追踪了现有的用于安装磷酸丝氨酸、磷酸苏氨酸、磷酸酪氨酸及其模拟物的 GCE 技术的发展，讨论了它们的优点和局限性。虽然有些技术仍处于开发早期，但其他技术已经足够强大，可以大大扩展可以解决的生物学相关问题的范围。我们重点介绍这些 GCE 方法带来的新发现，为非 GCE 专家应用技术提供实际考虑，并确定进一步发展的成熟途径。