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Applied and Environmental Microbiology, November 2007, p. 6714-6721, Vol. 73, No. 21
0099-2240/07/$08.00+0     doi:10.1128/AEM.01432-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

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

John Löfblom, Julia Sandberg, Henrik Wernérus, and Stefan Ståhl*

Department of Molecular Biotechnology, School of Biotechnology, Royal Institute of Technology, AlbaNova University Center, SE-106 91 Stockholm, Sweden

Received 27 June 2007/ Accepted 22 August 2007

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 (KD) determinations as well as dissociation rate analysis were performed using both methods, and the results show that the on-cell determinations give both KD 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.

* Corresponding author. Mailing address: School of Biotechnology, Royal Institute of Technology, AlbaNova University Center, SE-106 91 Stockholm, Sweden. Phone: 46 8 5537 8329. Fax: 46 8 5537 8481. E-mail: stefans{at}biotech.kth.se

{triangledown} Published ahead of print on 14 September 2007.

Applied and Environmental Microbiology, November 2007, p. 6714-6721, Vol. 73, No. 21
0099-2240/07/$08.00+0     doi:10.1128/AEM.01432-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.





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