Selection of Affibody Molecules Using Staphylococcal Display

Abstract

Affibody molecules are small (6-kDa) affinity proteins generated by directed evolution for specific binding to various target molecules. The first step in this workflow involves the generation of an affibody library, which can then be used for biopanning using multiple display methods. This protocol describes selection from affibody libraries using display on Staphylococcus carnosus. Display of affibodies on staphylococci is very efficient and straightforward because of the single cell membrane and the use of a construct with a constitutive promoter. The workflow involves display of affibody libraries on the surface of S. carnosus cells, followed by screening and selection of binders using fluorescence-activated cell sorting (FACS). The transformation of DNA libraries into S. carnosus is less efficient and more complicated than for Escherichia coli. Because of this, staphylococcal display is suitable for affinity maturation or other protein-engineering efforts that are not dependent on very high diversity, and thus magnetic-activated cell sorting (MACS) is often not required before FACS. However, MACS is an option, and MACS procedures used for E. coli can easily be adapted for use in S. carnosus if needed.