SBIR Phase I: Computer-Directed High Throughput Screening for Improved Enzymatic Activity

这个小企业创新研究第一阶段项目将开发一种计算机指导的高通量蛋白质筛选技术，以提高蛋白质的性能。通过将快速计算机筛选与实验方法相结合，可以大大扩展随机库筛选的能力。该技术将在环状芽孢杆菌木聚糖酶上进行测试，目的是提高其在高温和ph下的活性。从酶的晶体结构开始，Xencor专有的蛋白质设计自动化（PDA）技术将用于计算预筛选不同选择残基位置产生的可能序列，从而消除不利的序列，从而大大减少实验筛选的数量。计算机屏幕的结果是所选残基位置的氨基酸概率分布。将开发一种方法将这些概率转化为DNA文库，该文库将被合成并通过实验筛选以提高酶活性。与传统体外进化技术的10^10到10^15个序列相比，计算预筛选将允许筛选~10^80个序列。拟议的研究将导致一个普遍适用的程序，允许优化更多的残基位置，并通过耦合合理和随机方法更有效地开发改进的蛋白质。

This Small Business Innovation Research Phase I project will develop an enabling technology for computer-directed high throughput screening of proteins for improved properties. By combining a fast computer screen with experimental methods, the capabilities of random library screening can be greatly extended. This technology will be tested on Bacillus circulans xylanase with the goal of improving its activity at high temperature and pH. Starting from the enzyme crystal structure, Xencor's proprietary protein design automation (PDA) technology will be used to computationally prescreen the possible sequences resulting from varying selected residue positions, thereby eliminating unfavorable sequences and thus enormously reducing the number to be screened experimentally. The result of the computer screen is an amino acid probabilitydistribution for the selected residue positions. A method will be developed to transform these probabilities into a DNA library that will be synthesized and experimentally screened for improved enzymatic activity. The computational prescreening will allow screening of ~10^80 sequences compared to 10^10 to 10^15 with conventional in vitro evolution techniques. The proposed research will result in a generally applicableprocedure that allows optimization of far more residue positions and results in the more efficient development of improved proteins by coupling rational and random methods.