DNA files of all substrate proteins produced and described in Ryan et al., "A signaling inspired synthetic toolkit for efficient production of tyrosine phosphorylated proteins"

Tyrosine phosphorylation is an important post-translational modification that regulates many biochemical signaling networks in multicellular organisms. To date, 46,000 tyrosines have been observed in human proteins, but relatively little is known about the function and regulation of most of these sites. A major challenge has been producing recombinant phosphoproteins in order to test the effects of phosphorylation. Mutagenesis to acidic amino acids often fails to replicate the size and charge of a phosphorylated tyrosine residue and synthetic amino acid incorporation has high cost with relatively low yield. Here, we demonstrate an approach, inspired by how native tyrosine kinases find targets in cells -- through a secondary targeting interaction, augmenting innate catalytic specificity of a tyrosine kinase, without overriding it. We engineered complementary vector systems for multiple approaches to producing high yields of phosphoprotein products in E. coli. Here, we test phosphorylation as a function of the targeting interaction (an SH3-polyproline sequence) affinity, different reaction methods across kinases of different specificity. This system presents an inexpensive and tractable system to producing phosphoproteins and phosphopeptides and we demonstrate how it can be used for testing antibody specificity on targets of EGFR and PD-1. This methodology is a generalizable approach for enhancing the enzymatic action on a recombinant protein via the flexibility of in vitro reactions and co-expression approaches. We refer to this as SISA-KiT, for Signaling Inspired Synthetically Augmented Kinase Toolkit. <br>Described in detail at "A signaling inspired synthetic toolkit for efficient production of tyrosine phosphorylated proteins" Margaret M. Ryan, Reagan Portelance, Graham F. Newman, Gabrielle Martinez, Swathi Shekharan, Anqi Wu, Savannah Angel, Katherine E. Schaberg, Petra Gilmore, Robert Sprung, Reid Townsend, Kristen M. Naegle bioRxiv 2024.12.22.629992; doi: https://doi.org/10.1101/2024.12.22.629992

酪氨酸磷酸化（tyrosine phosphorylation）是一类重要的翻译后修饰（post-translational modification），可调控多细胞生物体内的诸多生物化学信号网络。截至目前，人类蛋白质中已发现46000个酪氨酸位点，但绝大多数这类位点的功能与调控机制仍鲜为人知。此前领域内的一大核心挑战在于获取重组磷酸化蛋白，以验证磷酸化修饰的生物学效应：将酪氨酸残基突变为酸性氨基酸往往无法精准模拟磷酸化酪氨酸的空间体积与电荷特征；而合成氨基酸掺入法不仅成本高昂，且产率相对较低。本研究受天然酪氨酸激酶在细胞内识别靶标的机制启发——通过次级靶向相互作用，在不改变酪氨酸激酶固有催化特异性的前提下增强其催化活性。我们构建了适配多种策略的配套载体系统，可在大肠杆菌（E. coli）中高效制备磷酸化蛋白产物。本研究以SH3-多脯氨酸序列的亲和力、不同特异性激酶的反应体系差异为变量，验证了靶向相互作用对磷酸化反应的调控作用。该系统可实现低成本且易操作的磷酸化蛋白与磷酸化肽制备，我们还展示了其可用于检测表皮生长因子受体（EGFR）与程序性死亡受体1（PD-1）靶标对应的抗体特异性。该方法具备通用性，可通过体外反应与共表达策略的灵活性，增强重组蛋白的酶促修饰效果，我们将其命名为SISA-KiT，即信号启发式合成增强激酶工具包（Signaling Inspired Synthetically Augmented Kinase Toolkit）。
相关研究详情见论文《高效制备酪氨酸磷酸化蛋白的信号启发式合成工具箱》（A signaling inspired synthetic toolkit for efficient production of tyrosine phosphorylated proteins），作者为Margaret M. Ryan、Reagan Portelance、Graham F. Newman、Gabrielle Martinez、Swathi Shekharan、Anqi Wu、Savannah Angel、Katherine E. Schaberg、Petra Gilmore、Robert Sprung、Reid Townsend、Kristen M. Naegle，发表于预印本平台bioRxiv 2024.12.22.629992；DOI: https://doi.org/10.1101/2024.12.22.629992