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

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 Impact

The 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.