Evaluation of Staphylococcal Cell Surface Display and Flow Cytometry for Postselectional Characterization of Affinity Proteins in Combinatorial Protein Engineering Applications

Abstract

For efficient generation of high-affinity protein-based binding molecules, fast and reliable downstream characterization platforms are needed. In this work, we have explored the use of staphylococcal cell surface display together with flow cytometry for affinity characterization of candidate affibody molecules directly on the cell surface. A model system comprising three closely related affibody molecules with different affinities for immunoglobulin G and an albumin binding domain with affinity for human serum albumin was used to investigate advantages and differences compared to biosensor technology in a side-by-side manner. Equilibrium dissociation constant (K D ) determinations as well as dissociation rate analysis were performed using both methods, and the results show that the on-cell determinations give both K D and dissociation rate values in a very fast and reproducible manner and that the relative affinities are very similar to the biosensor results. Interestingly, the results also show that there are differences between the absolute affinities determined with the two different technologies, and possible explanations for this are discussed. This work demonstrates the advantages of cell surface display for directed evolution of affinity proteins in terms of fast postselectional, on-cell characterization of candidate clones without the need for subcloning and subsequent protein expression and purification but also demonstrates that it is important to be aware that absolute affinities determined using different methods often vary substantially and that such comparisons therefore could be difficult.

Multiplex epitope mapping using bacterial surface display reveals both linear and conformational epitopes

“…Cell-surface display-based binding assays utilizing fluorescence detection are sensitive and agree with other biophysical characterization methods such as surface-plasmon resonance27. Using flow cytometry of S.carnosus -presented proteins, interactions of K D = 1.5 µM are clearly visible above background28.…”

Surface display of Salmonella epitopes in Escherichia coli and Staphylococcus carnosus

“…The procedure for flow cytometry analysis has been described before [19]. The proteins were visualised by fluorescence markers either to the E. coli His 6 tag or to the albumin-binding domain (ABD) in S. carnosus .…”

In vitro affinity screening of protein and peptide binders by megavalent bead surface display

“…When the display constructs contain a larger number of proteins—e.g. ∼10 4 copies displayed on bacteria (Chen et al , 1996; Andreoni et al , 1997; Christmann et al , 2001; Löfblom et al , 2005, 2007; Rockberg et al , 2008) or 30 000 copies on yeast (Boder and Wittrup, 1997)—selections can be based on the measurement of the binding property of every clone. Here, flow cytometry is employed to rank and sort binders.…”