Discipline: Biological Sciences
Subcategory: Biochemistry (not Cell and Molecular Biology and Genetics)
Brandy R. White - California State University, Fresno
Co-Author(s): Candace Bever, J. Gee, and Bruce D. Hammock, University of California, Davis, CA Cory L. Brooks, California State University, Fresno, CA
Brominated chemical flame retardants (BCFRs) have been used extensively in recent decades in furniture, plastics, wiring, and electronics because they were thought to prevent fires in homes and workplaces. However, there is growing concern of these BCFRs in living spaces as there is evidence of bioaccumulation in human blood, breast milk, and body fat, and while they mimic the endocrine system in rat models, they also have high potential of being carcinogenic. While BCFRs have recently been placed on the California Biomonitoring Program and have been banned by the EPA, these BCFRs persist in the environment. A detection method has been developed using a camelid variable heavy chain domain antibody (VHH)-based method of two of these BCFRs: 2,2’,4,4’-tetrabrominated diphenyl ether (BDE47) and Tetrabromobisphenol A (TBBPA). However, it is not well understood how these detection methods work and it is difficult to improve on the methods without this understanding. Camelid VHH have many advantages compared to conventional monoclonal antibodies including high stability and ease of recombinant production. Although it is well established how VHH interact with proteins, it is less well understood how these antibodies interact with small molecules and haptens. There are currently four known binding modes available for VHH to interact with small molecules including: combining VHH’s three CDR loops, domain swapping utilizing a 2:1 binding stoichiometry, and tunneling under the CDR1 loop. Using VHH specific for PBFRs as a model, we aim to examine VHH-small molecule interactions. We have successfully sequenced and overexpressed three anti-BDE47 and one anti-TBBPA proteins, as visualized with SDS-PAGE and size-exclusion chromatography. Crystallization trials of the four proteins have yielded preliminary crystals and our current focus is on crystal optimization. Additionally, physical properties of these VHH have been observed, including cold and light sensitivity. We anticipate this study to allow visualization of VHH-small molecule binding and that this will contribute to the bioengineering of future VHH-detection methods.
Funder Acknowledgement(s): Howell-CSUPERB Research Scholars Award; CSUF-ASI (FSSRA); CSUF-rGrant; Howard P Kubo MD Award; Helen Gigliotti Biochemistry Scholarship; Carl E Levin Science & Math Scholarship
Faculty Advisor: Cory L. Brooks, email@example.com
Role: Transformation of seven plasmids into three E.coli cell lines; expression tests; overexpression and purification of four nanobodies; SDS-PAGE gel electrophoresis; size exclusion chromatography; concentration of nanobodies in solution; plasmid purification for sequencing; robot trials; crystal optimization trials; enzymatic histag cleavage tests; monitoring for crystals; crystal extraction and freezing of crystals; data analysis.