Carbohydrate chains, known as glycans, are involved in many functions of a cell since they determine the properties of macromolecules such as proteins. Unfortunately it is difficult to determine the type and structure of glycans. However, antibodies that recognize and bind to specific glycans could offer a powerful tool for the analysis of these carbohydrates. The objective of this proposal is to create a robust method to generate glycan recognizing antibodies that will bind to specific chains and that can be used to analyze these structures. A better understanding of the role that glycans play will contribute to the fundamental knowledge of the behavior of cells which is critical for finding cures for diseases and for engineering efficient cells in biomanufacturing.
The objective of this proposal is to create a robust, integrated pipeline for the rapid discovery and characterization of selective, high-affinity Abs against defined glycan structures or glycan-polypeptide determinants of biomedical importance. The underlying hypothesis is that glycoengineered Escherichia coli can be leveraged for facile production of large quantities of relatively pure glycotopes, which can be efficiently immobilized to solid supports and used for parallel selection of glycobodies from phage-displayed synthetic Ab libraries. To test this hypothesis, an array of ~100 structurally uniform glycotopes will be synthesized using engineered bacteria carrying synthetic pathways of glycosyltransferases. Then, using a quasi-manual protocol, synthetic glycobodies against these glycotopes will be selected from large combinatorial libraries by iterative rounds of Fab-phage display. Finally, isolated glycobodies will be characterized for glycotope affinity and specificity using ELISA, surface plasmon resonance (SPR), and glycan microarrays. To establish biological relevance, glycobodies will be used to probe the expression of authentic glycan signatures on primary cell lines, viruses, or pathogenic bacteria. By coupling glyco-antigen expression (Aim 1) to antibody selection (Aim 2) within the same laboratory, this project is anticipated to yield an integrated pipeline for routine generation of orthogonal glycobody-glycotope pairs in a matter of weeks.
This award by the Biotechnology and Biochemical Engineering Program of the CBET Division is cosponsored by the Biomaterials Program of the Division of Materials Research.
