STABLE FOLD SELECTION FOR HETEROLOGOUS PROTEINS

异源蛋白质的稳定折叠选择

• 批准号: 6209153
• 负责人: ROBERT F BALINT
• 金额: $ 37.5万
• 依托单位: PANORAMA RESEARCH, INC.
• 依托单位国家: 美国
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-09-01 至 2002-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6209153
• 关键词: Escherichia coli; aminohydrolases; biotechnology; chemical stability; conformation; endopeptidases; horseradish peroxidase; method development; mutant; peptide library; protein engineering; protein folding; protein sequence; recombinant proteins

We have devised a novel solution to two related problems in protein engineering. (1) Production costs for many proteins of industrial or pharmaceutical importance are prohibitive due to sub-optimal expression of the recombinant protein in heterologous hosts. (2) Most cDNA expression libraries are so full of non-expressible sequences that the frequencies of many desired sequences may be inaccessible to available screening systems. This problem is particularly acute for high-throughput library screening, which will be essential to accelerate pharmaceutical target discovery and validation in the post-genomics era. In Phase I the method was used to improve the expression of the Green Fluorescent Protein (GFP) of Aequorea Victoria by a factor of 30 over the wild-type protein. A mutagenic library of GFP was expressed in E. coli as fusions with chloramphenicol (cam) acetyl transferase (CAT), and was selected for increased cam resistance. In addition, a single mutation was identified which specifically improved the expression of GFP in fusions with other proteins by a factor of 56 over wild-type. In Phase II the method will be used to optimize bacterial expression of several proteins of industrial or pharmaceutical value. The method will also be used to enrich expressed sequence libraries for autonomously folding domains. PROPOSED COMMERCIAL APPLICATION: The primary goal of this work is to develop new methods for engineering production values and stability in proteins of industrial and phamaceutical importance.

我们为蛋白质工程中的两个相关问题设计了一种新的解决方案。(1)由于重组蛋白在异源宿主中的次优表达，许多具有工业或医药重要性的蛋白质的生产成本是昂贵的。(2)大多数表达文库都含有大量的非表达序列，因此现有的筛选系统可能无法获得许多所需序列的频率。这个问题对于高通量文库筛选尤其严重，这将是后基因组时代加快药物靶点发现和验证的关键。在第一阶段，该方法被用来提高Aequorea Victoria绿色荧光蛋白(GFP)的表达水平，比野生型蛋白提高30倍。将GFP突变文库与氯霉素(CaM)乙酰转移酶(CAT)融合后在大肠杆菌中表达，筛选出对CaM抗性增强的GFP突变文库。此外，还发现了一个突变，该突变可以特异性地提高GFP与其他蛋白质融合时的表达，比野生型提高56倍。在第二阶段，该方法将用于优化几种具有工业或医药价值的蛋白质的细菌表达。该方法还将用于丰富自主折叠结构域的表达序列文库。拟议的商业应用：这项工作的主要目标是开发新的方法，以提高具有工业和医药重要性的蛋白质的工程生产价值和稳定性。