Protein Stability and Functionality Enhancement Method

描述（由申请人提供）：本研究的长期目标是开发称为插入、缺失、取代随机诱变（ids.PCR）的定向进化平台技术，用于诊断的商业应用。我们建议开发用于诊断和治疗监测的诊断报告酶，其需要对复杂混合物中存在的靶蛋白进行灵敏、特异、定量和多重检测。我们将产生β-内酰胺酶变体，其含有插入的氨基酸序列，对特定疾病相关靶蛋白具有结合亲和力。这些功能增强的“诊断报告酶”将联合收割机将β-内酰胺酶的高效催化活性与特异性抗体样结合亲和力结合，导致快速、特异、选择性和灵敏地检测与疾病相关的生物标记。对于这项II期研究，将使用两种商业相关的蛋白质靶点来筛选和表征进化的酶，以单独和复杂的患者样本对靶点作出反应。 建议的商业应用：Ids.PCR是一种平台进化技术，具有深远的潜在商业应用。使用该技术发展的诊断报告酶的示范将在诊断和治疗监测中具有广泛的应用。我们的目标是最终使用这些低成本的单分子试剂来筛选大量的生物标志物，用于分析复杂患者样本中的人类疾病。

DESCRIPTION (provided by applicant): The long range goal of this research is to develop the directed evolution platform technology termed Insertion, Deletion, Substitution random mutagenesis (ids.PCR) for commercial applications in diagnostics. We propose to develop diagnostic reporter enzymes for diagnosis and therapeutic monitoring that require sensitive, specific, quantitative and multiplexed detection of target proteins present in complex mixtures. We will generate beta-lactamase variants that contain inserted amino acid sequences with binding affinity for specific disease-associated target proteins. These functionally enhanced "Diagnostic Reporter Enzymes" will combine the highly efficient catalytic activity of beta-lactamase with specific antibody-like binding affinities, resulting in rapid, specific, selective, and sensitive detection of biological markers associated with disease. For this Phase II research, two commercially relevant protein targets will be used to screen and characterize the evolved enzymes for response to the target alone and in complex patient samples. PROPOSED COMMERCIAL APPLICATIONS: Ids.PCR is a platform evolution technology that has far-reaching potential commercial applications. The demonstration of diagnostic reporter enzymes evolved using this technology would have widespread application in diagnostics and therapeutic monitoring. The goal is to ultimately use these low-cost single molecule reagents to screen large numbers of biological markers for analysis of human diseases in complex patient samples.