Yeast Display Screening for Single Domain Antibody (sdAb)
Single domain antibodies (sdAbs) refer to the 15-kDa immunoglobulin VHH domain derived from camelids, sharks, or the specific developed human VH and VL domain. With the remarkable characterizations compared to conventional antibody, sdAbs present great potential as biochemical tools and therapeutic agents. Here, Creative Biolabs describes the novel yeast display screening for sdAb generation.
Background of Yeast Display
With the exceptional specificity and biochemical versatility, antibodies present a transformative impact in the field of medicine and science. As the most unique existence, sdAbs showed more powerful functions compared to conventional antibodies and fragments.
However, almost all the sdAbs available were obtained by animals (e.g. sharks, camelids) immunization, which is a lengthy process. What’s more, immune-based sdAbs are frequently restricted from binding conserved epitopes because of the immunological tolerance of self-antigens. A combination of phage display and the synthetic library has been used to solve this problem, but it is also difficult to obtain sdAbs bind both target and defined conformation. Here, the yeast display-based in vitro platform has been developed to address these challenges.
Fig.1 Design and construction of synthetic sdAb library. (McMahon, et al., 2018)
Yeast Display Screening
Based on the consensus framework derived from llama genes, the synthetic sdAb library can be constructed via partial randomization. The complementarity-determining loops (CDRs) contain the highly variable antigen-binding interface of the VHH. The library frequencies can be further analyzed by next-generation sequencing (NGS). Yeast-displayed sdAbs carry an HA tag at the carboxyl terminus, followed by a long, flexible stem that covalently attaches the sdAbs to the yeast cell wall.
Fig.2 Schematic of sdAb display on yeast and selection process. (McMahon, et al., 2018)
For target-specific sdAbs identification, the magnetic cell sorting is used. This approach entails the incubation of fluorescent target protein with synthetic yeast sdAb library. Labeled cells can then be isolated by magnet-based separation and amplified in the culture medium. At first, the fluorescently labeled target protein is prepared to perform iterative staining and magnetic-bead-based enrichments. To make sure that the selected binders recognize the target protein rather than fluorescent tags or reagents, the beads-specific yeast were depleted from the library before each round of screening. What’s more, different fluorophore labels are used alternately in each successive magnetic selection round to decrease the probability of enriching fluorophore binders. In general, after four rounds of screening, the single yeast colonies can be obtained and validated by flow cytometry.
Reference
- McMahon, C.; et al. Yeast surface display platform for rapid discovery of conformationally selective nanobodies. Nature structural & molecular biology. 2018, 25(3): 289-296.
Case Study
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