Figure 1

Schematic illustration of the expression cassette, including the recombinant protein to be displayed on E. coli as well as the optional process for selection from displayed libraries or secreted production. A. The expression cassette (EC) contains a promoter region, a signal peptide for translocation over the inner membrane, an Affibody molecule as binding protein, a His-tag for purification, an OmpT cleavage site for secretion, a surface expression reporter-tag for normalization and the AIDA-C for insertion into the outer membrane. AIDA-I numbering is according to the UniProt accession number Q03155 (http://www.uniprot.org). B. The recombinant fusion protein expressed in two different strains of E. coli. In an OmpT negative strain the recombinant protein remains tethered in the outer membrane and thus displayed on the bacterial surface, allowing for phenotype-genotype linkage and FACS. In an OmpT positive strain, OmpT will cleave the recombinant protein and release the Affibody molecule fused to a His-tag into the medium. C. Schematic drawing of a potential workflow using E. coli display for selecting Affibody molecules from libraries using FACS. After selection of new Affibody variants, the expression vector is transformed into an OmpT positive strain that results in protease-mediated secretion of candidates for downstream characterization.