Abstract
Fusions to the C-terminal end of the Aga2p mating adhesion of Saccharomyces cerevisiae have been used in many studies for the selection of affinity reagents by yeast display followed by flow cytometric analysis. Here we present an improved yeast display system for the screening of Nanobody immune libraries where we fused the Nanobody to the N-terminal end of Aga2p to avoid steric hindrance between the fused Nanobody and the antigen. Moreover, the display level of a cloned Nanobody on the surface of an individual yeast cell can be monitored through a covalent fluorophore that is attached in a single enzymatic step to an orthogonal acyl carrier protein (ACP). Additionally, the displayed Nanobody can be easily released from the yeast surface and immobilised on solid surfaces for rapid analysis. To prove the generic nature of this novel Nanobody discovery platform, we conveniently selected Nanobodies against three different antigens, including two membrane proteins.
Highlights
The need for specific affinity reagents has steadily been growing to fulfil the increasing needs in diagnosis, imaging and proteomics[1]
The display level of a cloned Nanobody on the surface of an individual yeast cell can be monitored through a covalent fluorophore that is attached in a single enzymatic step to an orthogonal acyl carrier protein (ACP) tag[35]
Coenzyme A (CoA) derivatives, including fluorescent or biotinylated variants can be enzymatically coupled through a covalent bond to a conserved serine residue by the phosphopantetheinyl transferases from Escherichia coli (AcpS) or Bacillus subtilis (Sfp) (Fig. 2b)
Summary
The need for specific affinity reagents has steadily been growing to fulfil the increasing needs in diagnosis, imaging and proteomics[1]. Nanobodies are the small (15 kDa) and stable single-domain fragments of the naturally occurring heavy chain-only antibodies, found in camelids[4] This special IgG subclass is capable of binding to common antigenic determinants, protein flat surfaces[5,6], peptides[7] and small haptens[8,9] comparably to conventional antibodies. The major advantage of cell-surface display is the compatibility of these methods with the quantitative and multi-parameter analysis offered by flow cytometry[24] In this connection, each individual cell of the library can be investigated one by one for the display level of the cloned affinity reagent and its antigen occupancy in real time[18], under well-controlled conditions including buffer composition, pH, temperature and antigen concentration.
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