title: Single-cell characterization of autotransporter-mediated Escherichia coli surface display of disulfide bond-containing proteins.
abstract: Autotransporters (ATs) are a family of bacterial proteins containing a C-terminal β-barrel-forming domain that facilitates the translocation of N-terminal passenger domain whose functions range from adhesion to proteolysis. Genetic replacement of the native passenger domain with heterologous proteins is an attractive strategy not only for applications such as biocatalysis, live-cell vaccines, and protein engineering but also for gaining mechanistic insights toward understanding AT translocation. The ability of ATs to efficiently display functional recombinant proteins containing multiple disulfides has remained largely controversial. By employing high-throughput single-cell flow cytometry, we have systematically investigated the ability of the Escherichia coli AT Antigen 43 (Ag43) to display two different recombinant reporter proteins, a single-chain antibody (M18 scFv) that contains two disulfides and chymotrypsin that contains four disulfides, by varying the signal peptide and deleting the different domains of the native protein. Our results indicate that only the C-terminal β-barrel and the threaded α-helix are essential for efficient surface display of functional recombinant proteins containing multiple disulfides. These results imply that there are no inherent constraints for functional translocation and display of disulfide bond-containing proteins mediated by the AT system and should open new avenues for protein display and engineering.
authors.affiliation: Department of Chemical and Biomolecular Engineering, University of Houston, Houston, Texas 77204, USA.
authors.forename: Balakrishnan
authors.initials: B
authors.lastname: Ramesh
authors.forename: Victor G
authors.initials: VG
authors.lastname: Sendra
authors.forename: Patrick C
authors.initials: PC
authors.lastname: Cirino
authors.forename: Navin
authors.initials: N
authors.lastname: Varadarajan
mesh_terms: D000818:Animals; D001692:Biological Transport; D002462:Cell Membrane / Q000378:metabolism*; D002918:Chymotrypsin / Q000737:chemistry; D003001:Cloning, Molecular; D004220:Disulfides / Q000737:chemistry; D004926:Escherichia coli / Q000378:metabolism*; D005434:Flow Cytometry / Q000379:methods; D007700:Kinetics; D010455:Peptides / Q000737:chemistry; D010957:Plasmids / Q000378:metabolism; D015202:Protein Engineering / Q000379:methods; D017433:Protein Structure, Secondary; D021381:Protein Transport; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry; D057057:Serine Proteases / Q000737:chemistry; D013499:Surface Properties
chemical_list: D004220:Disulfides; D010455:Peptides; D011994:Recombinant Proteins; D057057:Serine Proteases; D002918:Chymotrypsin