Biomolecular scaffolds for enhanced biomass processing and tumor marker detection

Date
2015
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University of Delaware
Abstract
Proteins, as key components of life, are not standalone pieces. They frequently work cohesively either to form complex multi-step biochemical reactions in metabolic process or signaling pathway to receive specific inputs/signals and translate them into specified output responses. With all these protein collaborating to sustain life, nature has evolved protein co-localization on biomolecular scaffolds to enhance pathway efficiency. Inspired by the remarkable and extensive advantages provided by these scaffolds, we organized proteins on synthetic nucleotide or protein scaffolds to create protein co-localization systems for cellulose hydrolysis and cancer marker detection. In objective one, we focused on organizing cellulases on DNA scaffolds to construct artificial cellulosomes based on zinc finger protein (ZFP)-guided assembly. Cellulosomes are naturally occurring multi-enzyme complexes with key components of cellulose binding module (CBM) and cellulases. Although artificial cellulosomes on protein scaffolds with up to 6 enzymes have been constructed with enhanced behavior, extending artificial cellulosome to more complex structures on protein scaffolds is still challenging as multi-domain large proteins tend to aggregate due to incorrect folding. Focusing on solving this problem, DNA templates were picked as scaffolds with the advantages of flexibility, easy synthesis and readily available complex structures. Zinc finger proteins as the DNA binding proteins were utilized to enable chemical modification free protein organization for artificial cellulosome assembly. Our second objective was to improve the performance of DNA scaffold-based artificial cellulosomes by having a more stable immobilization. To achieve that, a xv mutant dehalogenase enzyme (HaloTag) was used to replace the ZFP, such that fusion proteins can be covalently attached onto DNA linker, and DNA hybridization was used to immobilize cellulosome components onto a DNA scaffold. This alteration resulted in a system with increased enzyme efficiency compared with zinc finger protein-based assembly. A complex artificial cellulosome on a rolling circle amplification DNA template was achieved with 5-fold enhancement of cellulose hydrolysis efficiency. Our final objective was to utilize three dimensional protein nanoparticles as platform for biosensor assembly for antigen and cancer cell detection. Protein-based nanoparticles have emerged as an excellent platform for biosensor assembly; however, current strategies of decorating bionanoparticles with different sensing and detection moieties often suffer from unfavorable spacing and orientation as well as bionanoparticle aggregation. To solve these problems, we use a highly modular post-translational modification approach, which enables the simultaneous modification of the Bacillus stearothermophilus E2 nanoparticles with different functional moieties for antibody, enzyme, DNA and dye decoration. The resulting platform offers easy purification, signal amplification and a high degree of targeting and sensing modularity. These advantages are demonstrated by the detection of both immobilized antigens and cancer cells.
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