The aim of the study presented herein was the development and examination of a system for the non-covalent display of antibodies and antibody-derived molecules as well as libraries thereof on the surface of yeast cells as a technology for drug discovery.
The method of non-covalent way surface display enables the selection of specific variants via high-throughput-screening and the subsequent switchable secretion of the selected binder into the supernatant for production and biochemical characterization without changing the expression host. The key-point is the successful combination of selection and production to circumvent time-consuming sub-cloning steps that are necessary within the traditional technologies. Due to the utilization of a Fc-binding domain as a mediator of non-covalent display of IgG and Fc-fusion molecules it is possible to selectively switch between surface display and soluble secretion. This can be achieved by an inducible expression mode that can easily be switched between membrane-bound and soluble expression. The main advantage of this approach is the display, selection and production of these proteins in their final format, which ensures reproducible protein-features within each of the before-mentioned steps.
The key-issue in this work was to verify a stable genotype/phenotype-coupling between displayed protein variant and cellular genetic information which is a requirement for successful selection strategies. This was achieved by successfully conducting several spiking experiments. Furthermore different VHH-libraries were created with common in vitro and in vivo diversification technologies and screened within this new yeast display platform for variants with desired properties and needs by utilizing MACS and FACS.
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