Protein Stability and Functionality Enhancement Method

蛋白质稳定性和功能增强方法

• 批准号: 7489256
• 负责人: Eric Lietz
• 金额: $ 37.58万
• 依托单位: GENOPSYS, INC.
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-03-01 至 2008-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7489256
• 关键词: Affinity; Amino Acid Sequence; Antibodies; Bacillus licheniformis; Binding; Binding Sites; Biological Assay; Biological Markers; Cell surface; Characteristics; Clinical; Complex; Complex Mixtures; DNA; DNA Insertion Elements; DNA Sequence; Detection; Diagnosis; Diagnostic; Diffuse; Disease; ERBB2 gene; Enzymes; Evolution; Gel; Generations; Genes; Goals; Grant; HercepTest; Immunohistochemistry; Insertion Mutation; Lactamase; Libraries; Location; Marketing; Membrane; Methods; Monitor; Mutagenesis; Numbers; Patients; Phase; Polymerase Chain Reaction; Polynucleotides; Progress Reports; Property; Proteins; Randomized; Range; Reagent; Reporter; Research; Research Personnel; Sampling; Solid; System; Technology; Testing; Therapeutic; Translating; Variant; Vascular Endothelial Growth Factors; Work; base; beta-Lactamase; beta-Lactams; cancer cell; commercial application; commercialization; concept; cost; cytokine; desire; directed evolution; enzyme activity; high throughput screening; human disease; indigo dye; indoxyl; insertion/deletion mutation; novel; polyacrylamide; response; single molecule; size; transposon/insertion element; tumor

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.

本研究的长期目标是开发定向进化平台技术， 插入、缺失、取代随机诱变（ids.PCR）在诊断中的商业应用。 我们建议开发用于诊断和治疗监测的诊断报告酶， 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。 我们将产生含有插入的氨基酸序列的β-内酰胺酶变体，所述插入的氨基酸序列具有与 特异性疾病相关靶蛋白。这些功能增强的“诊断报告酶” 将β-内酰胺酶的高效催化活性与特异性抗体样结合联合收割机结合 亲和性，导致快速，特异性，选择性和敏感性检测与 疾病对于这项第二阶段研究，两个商业相关的蛋白质目标将用于筛选， 表征进化的酶对单独的靶标和复杂的患者样品的响应。 建议的商业应用：Ids.PCR是一种平台进化技术， 具有深远的商业应用潜力。诊断报告酶的发展证明 使用该技术将在诊断和治疗监测中具有广泛的应用。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， 分析复杂患者样本中的人类疾病。