CAREER:New Tools for High-Throughput Screening of Protein Libraries: Engineering Metalloproteins Displayed on Bacillus Subtilis Spores

酶的关键特征是底物识别和催化活性，了解这些属性将对社会产生广泛的影响。定向进化方法已成为理解结构/功能关系和改善蛋白质特性的关键蛋白质工程工具。目的是证明枯草芽孢杆菌孢子是有用的高通量筛选。在枯草芽孢杆菌表面的蛋白质展示比更常用的微生物细胞表面展示系统（包括革兰氏阴性菌、噬菌体和酵母）有优势。例如，避免了与表达的重组多肽穿过膜相关的蛋白质折叠问题。因此，可以探索以前无法进入的蛋白质空间的不同区域。此外，孢子可以忍受许多极端的物理/化学条件；因此，所展示的蛋白质被“预先固定”在固有惰性的孢子表面上。固定化蛋白质在工业过程中有几个优点。提高了蛋白质的稳定性，简化了分离。最后，固定化蛋白质可以用于广泛的简单设备应用和配置。阐明金属蛋白的结构/功能关系。最初，孢子展示将使用含铜酶CotA进行演示，这是存在于枯草芽孢杆菌孢子外壳上的。它将发展为增加氧化还原电位和底物选择性。该突变体将为纸张漂白和多环芳烃的生物修复提供绿色替代方案。最后，CotA系统适用于枯草芽孢杆菌孢子上的异源蛋白展示，为蛋白文库的筛选提供了依据。更广泛的影响该研究在分子进化的多学科领域培养本科生和研究生。新泽西理工学院化学与环境科学系正在为本科生和研究生开设一个新的生物化学项目，该项目由首席研究员和最近的新雇员组成。正在建立一个基础设施，提供生物化学、分子生物学和蛋白质工程方面的专业知识和资源。新的课程已经开发，以填补缺失的程序组件和引入新的主题。课程包括生物化学实验、分子生物技术、环境生物学和环境微生物学。低收入和第一代或代表性不足的本科生有机会通过NJIT的Ronald E. McNair项目贡献原创性研究。PI在罗格斯-纽瓦克大学生物科学系研究生院任职，该学院促进了新泽西理工大学化学和罗格斯-纽瓦克生物之间的多学科研究。这项研究也将通过新泽西理工大学预科项目中心向非技术观众进行介绍。高中学生有机会参加由新泽西理工大学赞助的化学奥林匹克比赛，经济困难的高中学生也可以通过SEED项目参与基础生物化学研究。此外，计划中的研究将加强国际伙伴关系。有趣的CotA变体的结构将与葡萄牙新里斯本大学的一个晶体学研究小组合作解决。

Key features of enzymes are substrate recognition and catalytic activity, and understanding these attributes would have a broad impact on society. Directed evolution methods have emerged as a key protein engineering tool for understanding structure/function relationships and improving protein properties. The goal is to demonstrate that Bacillus subtilis spores are useful for high-throughput screening. Protein display on the surface of B. subtilis offers advantages over more commonly utilized microbe cell-surface display systems, which include gram negative bacteria, phage, and yeast. For instance, protein folding problems associated with the expressed recombinant polypeptide crossing membranes are avoided. Hence, a different region of protein space can be explored that previously was not accessible. In addition, spores tolerate many physical/chemical extremes; therefore, the displayed proteins are "preimmobilized" on the inherently inert spore surface. Immobilized proteins have several advantages when used in industrial processes. The protein stability is increased and separations are simplified. Finally, immobilized proteins can be used in a wide array of simple device applications and configurations. Structure/function relationships will be elucidated for metalloproteins. Initially, spore display will be demonstrated using the copper containing enzyme CotA, which is present on the B. subtilis spore coat. It will be evolved for increased redox potential and substrate selectivity. The mutants will provide a green alternative for paper bleaching and bioremediation of polycyclic aromatic hydrocarbons. Finally, the CotA system can adapted for heterologous protein display on B. subtilis spores that will be valuable for screening protein libraries.Broader ImpactThe research trains undergraduate and graduate students in the multidisciplinary field of molecular evolution. The Department of Chemistry and Environmental Science at the New Jersey Institute of Technology is creating a new Biochemistry program at the undergraduate and graduate level and it is being built around the principal investigator and recent new hires. An infrastructure is being created providing expertise and resources in biochemistry, molecular biology, and protein engineering. New courses have been developed to fill missing program components and to introduce new topics. Courses include Biochemistry Lab, Molecular Biotechnology, Environmental Biology and Environmental Microbiology. Low-income and first-generation or underrepresented undergraduates have an opportunity to contribute original research through the Ronald E. McNair program at NJIT. The PI has an appointment in the Graduate Faculty in the Department of Biological Science at Rutgers-Newark, which fosters multidisciplinary research between NJIT Chemistry and Rutgers-Newark Biology. The research will also be described to a non-technical audience through the Center of Pre-College Programs at NJIT. High school students have an opportunity to compete in Chemistry Olympics, which is sponsored by NJIT, and economically disadvantaged high school students can also be involved in basic biochemistry research through Project SEED. Also, the planned research enhances international partnerships. The structures of the interesting CotA variants will be solved in collaboration with a crystallographic research group at the Universidade Nova de Lisboa in Portugal.