An engineered lipoprotein binding the Fc region of IgGs. In APEx, the outer membrane of E. coli is permeabilized and the generated Benzethonium Chloride spheroplasts are incubated with the antigen labeled with a fluorophore or biotin, and subsequently selected by fluorescence activated cell sorting. Although phage display and APEx are robust technologies for Ab selection, alternative methods that enable the direct display of Abs or Ab libraries on the surface of E. coli cells, without the need for generation of Phabs or spheroplasts would be of great interest. In addition, E. coli cell display would facilitate selections by cell sorting methods using antigen in solution as well as the analysis of the selected clones by flow cytometry. Alternative successful cell display technologies developed for Ab selection utilize yeasts and Grampositive bacteria. In these cell display systems, the Ab fragments translocate across a single cell membrane and are anchored in the cell wall. Nevertheless, E. coli remains a more suitable microorganism for the generation, amplification and maintenance of large Ab repertoires owing to its high-efficiency of transformation and versatile expression systems. Despite these advantages, the presence of the OM has hindered the development of effective E. coli cell display methods for Ab selection, with the exception of the use of the chimeric lipoprotein Lpp-OmpA’ for the display of scFvs and selection of variants with higher affinity after mutagenesis of the scFv. Lpp-OmpA’ consists of the Nterminal SP and first 9 residues of the mature Lpp fused to residues 46 to 159 of OmpA, which is a truncated fragment of its native 8-stranded ��-barrel. However, this chimeric construct lacks the characteristic stability of the ��-barrel of native OM proteins and its expression induces OM leakage as well as cellular toxicity, which may have limited its use to the affinity maturation of scFvs. Other OMPs have also been used to display heterologous peptides and proteins on the surface of E. coli cells. Among them, the autotransporters and Intimin/Invasin proteins are very attractive display systems. Interestingly, an AT protein has been used in E. coli for the display and affinity maturation of an Anticalin protein scaffold binding human cytotoxic T-lymphocyte antigen 4. Protein members of the AT and Int/Inv families are large, secreted polypeptides that contain three functional regions: i) a N-terminal SP, that drives their Sec-dependent translocation across the IM; ii) a ��-domain, 
that is anchored into the OM and comprises a 12-stranded ��-barrel with a peptide linker running through the lumen of the ��-barrel; and iii) a passenger region, that is secreted to the extracellular milieu. Although their mechanism of secretion remains uncertain, AT and Int/Inv proteins are translocated into the periplasm and then use the ��-barrel assembly machine complex for insertion into the OM and translocation of the passenger region to the cell surface. Despite their similarities, AT and Int/Inv proteins also have important differences. Firstly, they have opposite topological Dimesna organization in the OM, being the passenger region located in the N-terminal portion of ATs whereas in the case of Int/Inv proteins is found in the C-terminus. The distinct topologies are also reflected in their ��-domains. In the case of ATs, the ��-barrel is preceded by ��-helix linker that fills the lumen and connects its N-terminus with the passenger region. In contrast, the ��-barrel of Int/Inv proteins is followed by a peptide linker that runs through the lumen connecting its C-terminus to the passenger region.