Protease Engineering

具有量身定制的物理和催化特性的酶的设计是 现代蛋白质科学的基本推动力，具有深刻的潜力 技术和医疗影响。实验室指导的进化方法 通过合理的设计原理，现在已经成功地应用于增强蛋白质 属性和功能。剩下的是重要的下一步，“把我们的 并着眼于细节来解决重要问题，这可能会 是特定于酶的。这项提议将我们的酶定向进化计划扩展到 工程蛋白水解酶领域的重要赋能方向。我们最新的工作 到目前为止，OmpT蛋白水解酶代表了对P1和P1‘最普遍的操作 底物专一性，同时保持较高的整体催化活性水平。 除了在洗涤剂工业中的使用外，工程蛋白酶还具有巨大的 作为蛋白质组学工具或催化疗法的实用潜力。特别是， 含有磷酸化或O-修饰等修饰的底物特有的蛋白酶 GlcNAc将是鉴定高表达修饰蛋白质的有用新工具 蛋白质组学分析。在具体目标1下，我们将设计OmpT变体 专用于仅切割含有磷酸化或O-GlcNAc丝氨酸的底物。 我们还将研究我们是否能设计出类似限制的蛋白水解酶，从而 非常选择性地识别包含远超过P1的残基的延伸序列 P1‘。在特定目标2下，我们将扩展准确的OmpT蛋白酶识别，以包括 P2、P3、P2‘和P3’。特别是，我们将以Gln-His-Ala-Arg-Ala-Ser为目标 (QHA$RAS)，C3a过敏性毒素多肽C末端的68-73个残基。丙- C3a的末端裂解干扰了其在补体激活中的生物学作用。 我们认识到，通过突变和突变在OmpT中创建高度特异性的亚位点 分类是一个令人兴奋但有风险的目标，而设计一个OmpT是不太可能的 因其细菌来源而成为临床治疗候选药物。因此，为了特定的目的， 3，我们将在分泌的人胰酶样蛋白中设计出精确的C3a裂解活性 蛋白水解酶颗粒酶A，希望产生临床候选。作为一个实用的 可交付，根据我们最好的变种的功能分析(D6节)，我们将 第一次，能够验证蛋白质分解方法来抑制补体， 适用于多种炎症性疾病的治疗。

The design of enzymes with tailored physical and catalytic properties is one of the fundamental thrusts of modern protein science, with the potential for profound technological and medical impact. Laboratory directed evolutionary approaches along with rational design principles have now been successfully applied to enhance protein properties and function. What remains is the important next step of "rolling up our sleeves" and attacking important problems with an eye toward details, which are likely to be enzyme specific. This proposal extends our enzyme directed evolution program in important enabling directions in the area of engineered proteases. Our most recent work with the OmpT protease represents by far the most general manipulation of P1 and P1' substrate specificity of a protease while retaining high overall levels of catalytic activity. Beyond their use in the detergent industry, engineered proteases have tremendous practical potential as either proteomic tools or catalytic therapeutics. In particular, proteases specific for substrates containing modifications such as phosphorylation or O- GlcNAc would represent useful new tools for identifying modified proteins in high throughput proteomics assays. Under Specific Aim 1, we will engineer OmpT variants specific for cleaving only substrates containing phosphorylated or O-GlcNAc serine. We will also investigate whether we can engineer "restriction-like" proteases that can very selectively recognize an extended sequence comprising residues well beyond P1 and P1'. Under Specific Aim 2, we will extend precise OmpT protease recognition to include P2, P3, P2', and P3'. In particular, we will target Gln-His-Ala-Arg-Ala-Ser (QHA$RAS), residues 68-73 of the C-terminus of the C3a anaphylatoxin peptide. C- terminal cleavage of C3a interferes with its biological effects in complement activation. We recognize that creating highly specific subsites in OmpT by mutagenesis and sorting is an exciting yet risky goal and that an engineered OmpT is an unlikely therapeutic clinical candidate because of its bacterial origin. Therefore, for Specific Aim 3, we will engineer precise C3a cleavage activity into the secreted human trypsin-like protease granzyme A in hopes of producing a clinical candidate. As a practical deliverable, following the functional assays of our best variants (Section D6) we will, for the first time, be able to validate the proteolytic approach to complement inhibition, applicable to a wide variety of inflammatory disease therapies.