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

酶的主要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。定向进化方法已经成为理解结构/功能关系和改善蛋白质性质的关键蛋白质工程工具。目的是证明枯草芽胞杆菌芽胞在高通量筛选中是有用的。枯草杆菌表面的蛋白质展示系统比更常用的微生物细胞表面展示系统更具优势，这些微生物细胞表面展示系统包括革兰氏阴性细菌、噬菌体和酵母。例如，避免了与表达的重组多肽跨膜相关的蛋白质折叠问题。因此，可以探索蛋白质空间中以前无法访问的不同区域。此外，孢子耐受许多物理/化学极端；因此，所展示的蛋白质被预先固定在固有的惰性孢子表面。固定化蛋白质在工业过程中使用时有几个优点。提高了蛋白质的稳定性，简化了分离。最后，固定化蛋白可用于各种简单的设备应用和配置。金属蛋白的结构/功能关系将被阐明。最初，将使用枯草杆菌孢子衣上的含铜酶Cota展示孢子。它将为提高氧化还原电位和底物选择性而发展。这些突变体将为纸张漂白和多环芳烃的生物修复提供一种绿色替代方案。最后，Cota系统可以适应枯草杆菌孢子上的异源蛋白质展示，这将对筛选蛋白质文库有价值。布罗德影响这项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和环境科学系正在为本科生和研究生创建一个新的生物化学课程，该课程是围绕首席研究人员和最近招聘的新员工而建立的。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。已经开发了新的课程，以填补缺失的课程组成部分，并引入新的主题。课程包括生物化学实验室、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的罗纳德·E·麦克奈尔项目贡献原创研究。PI在罗格斯-纽瓦克大学生物科学系的研究生院有一个职位，该系促进NJIT化学和罗格斯-纽瓦克生物学之间的多学科研究。这项研究也将通过NJIT大学预科项目中心向非技术类受众介绍。高中生有机会参加由NJIT赞助的化学奥林匹克，经济困难的高中生也可以通过种子计划参与基础生物化学研究。此外，计划中的研究还加强了国际合作伙伴关系。有趣的Cota变体的结构将与葡萄牙里斯本大学的一个结晶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.