[30] Use of an Lpp-OmpA fusion vehicle for bacterial surface display

This chapter discusses the application of lipoprotein (Lpp)–OmpA fusion vehicle to study the surface of bacteria. The Lpp–OmpA vector is designed for use in the gram-negative bacterium Escherichia coli; the rationale for its dibrid form stems from the architecture of the E. coli envelope and the properties of the major Lpp and OmpA proteins. The envelope of gram-negative cells consists of (i) a cytoplasmic membrane, (ii) the periplasm, in which the peptidoglycan cell wall is located, and (iii) the outer membrane (OM), a unique structure composed of proteins embedded in an asymmetric lipid bilayer containing phospholipid in the inner leaflet and lipopolysaccharide (LPS) in the outer leaflet. In the Lpp-OmpA vector, the Lpp domain serves to target and anchor the tribrid to the OM and the OmpA domain is required for the surface expression of the passenger. The relevant properties of these two proteins follow immediately. The Lpp protein of E. coli is an OM protein and is, therefore, synthesized with a signal sequence and exported, numerically the most abundant protein of E. coli, present as a trimer, and is positioned in the OM such that most of the protein is located in the periplasm. A fusion protein consisting of the Lpp signal sequence and the first nine amino acids of mature Lpp linked to the normally soluble, periplasmic protein TEM β-lactamase (Bla) was localized to the OM. This ability of the Lpp amino terminus to target passenger proteins to the OM is utilized in the Lpp–OmpA construct.