FWO-SB-Beurs: Innovating Yeast Display for the selection of nanobodies for difficult targets

  • Steyaert, Jan (Administrative Promotor)
  • Uchanski, Tomasz, (Mandate)

Project Details

Description

For decades, antibodies have played a central role as research tools and more recently also as therapeutic agents (biologicals). Their impact on research and medicine is still growing. It was shown that Nanobodies (Nbs), small (15 kDa) and stable single-domain antibody fragments, can bind the most difficult targets including membrane proteins, transient multiprotein assemblies, intrinsically disordered proteins. In the Steyaert lab, a pioneer in Nb technology, phage and yeast display are the methods of choice. Phage display presents a robust and quick method to screen antibodies, however the selection experiments are performed blind. Yeast display on the other hand can be coupled with fluorescent-activated cell sorting (FACS) and through the resolving power of FACS each Nb population and individual clone selected can be characterized. Current yeast-display methods are limited by laborious, high-cost labeling steps and the success of performed selection relays on soluble fluorescent target proteins, problematic for membrane proteins and protein complexes. 
In order to overcome those issues, it is our ambition to develop innovative yeast display methods for the selection of Nbs allowing the structural and functional characterization of the most difficult (drug) targets. Aiming at improving the quality staining yeast cells expressing Nb we will develop orthogonal staining methods in which each Nb displayed on the yeast cell is covalently labeled with a fluorescent. By Developing Cell to Cell selection (C2C selection), membrane proteins will be targeted more efficiently even without protein purification steps. Utilization of Chill&Disco platformem will allow selection of Nanobodies stabilizing next difficult targets: transient protein-protein interactions. In this purpose ßarrestin interactome will be investigated. Successful development of those innovative methods will open new venues for the development of research tools and will also find applications in small-molecule drug discovery and the development of new biologicals. During presented project many technics will be used. Most of them are classified into groups, listed below: i) Protein engineering: designing of expression vectors, molecular cloning, mutagenesis, protein expression in prokaryotic systems, protein expression in eukaryotic systems (insect cells), protein purification (chromatography technics) , protein labeling ii)protein characterization: Enzyme-Linked Immunosorbent Assay (ELISA), Surface Plasmon Resonance (SPR), western blotting, radioligand assay iii) yeast display technology: yeast cells cultures, FACS (Fluorescence-activated cell sorting). The Steyaert Lab, as a part of Structural Biology Brussels group at VUB, fulfils all requirements, stated within the project. Therefore all experiments will be performed in the host lab. Considering the difficulties associated with membrane protein, these projects will be conducted in close collaboration with Robert Lefkowitz (Duke University) and Brian Kobilka (Stanford University) who are the world-leading experts in this field.
AcronymFWOSB2
StatusFinished
Effective start/end date1/01/1631/12/19

Keywords

  • Antibodies
  • Yeast
  • Nanobodies

Flemish discipline codes

  • Biology and other natural sciences