Autodisplay: one-component system for efficient surface display and release of soluble recombinant proteins from Escherichia coli

Abstract

The immunoglobulin A protease family of secreted proteins are derived from self-translocating polyprotein precursors which contain C-terminal domains promoting the translocation of the N-terminally attached passenger domains across gram-negative bacterial outer membranes. Computer predictions identified the C-terminal domain of the Escherichia coli adhesin involved in diffuse adherence (AIDA-I) as a member of the autotransporter family. A model of the ␤-barrel structure, proposed to be responsible for outer membrane translocation, served as a basis for the construction of fusion proteins containing heterologous passengers. Autotransporter-mediated surface display (autodisplay) was investigated for the cholera toxin B subunit and the peptide antigen tag PEYFK. Up to 5% of total cellular protein was detectable in the outer membrane as passenger autotransporter fusion protein synthesized under control of the constitutive P TK promoter. Efficient presentation of the passenger domains was demonstrated in the outer membrane protease T-deficient (ompT) strain E. coli UT5600 and the ompT dsbA double mutant JK321. Surface exposure was ascertained by enzyme-linked immunosorbent assay, immunofluorescence microscopy, and immunogold electron microscopy using antisera specific for the passenger domains. In strain UT2300 (ompT ؉ ), the passenger domains were released from the cell surface by the OmpT protease at a novel specific cleavage site, R2V. Autodisplay represents a useful tool for future protein translocation studies with interesting biotechnological possibilities.

Autodisplay: Functional Display of Active β-Lactamase on the Surface of Escherichia coli by the AIDA-I Autotransporter

“…The control construct (pLAT83) expressing wild-type Bla in the periplasm was obtained by the same strategy, replacing primer A3 with A2 (5Ј-GATCAGATCT AGATTACCAATGCTTAATCAGTG-3Ј), incorporating the stop codon of the bla gene. Plasmid pJM1013 is a medium-copy-number vector expressing the reporter epitope PEYFK fused to the AIDA-I autotransporter domain under the strong P TK promoter, similar to pJM22 (24). The expression of the AIDA-I fusion proteins was analyzed by the separation of the outer membrane fraction of E. coli by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) or by Western blotting using a rabbit antiserum raised against Bla.…”

“…In these studies, both autotransporter domains were shown to efficiently mediate the export of heterologous passenger proteins, such as the B subunit of cholera toxin (CTB), as well as defined epitopes, to the surfaces of E. coli and Salmonella enterica serovar Typhimurium cells. The amount of the heterologous passenger protein presented on the surface was up to 5% of the total bacterial protein in the AIDA-I system (24), demonstrating the potential of autotransporters for biotechnological applications. The autotransporter domains of the Serratia marcescens serine protease (35) and the Shigella flexneri VirG protein (38) have also been successfully employed for surface display.…”

Type V Protein Secretion Pathway: the Autotransporter Story

“…The seminal work on secretion has also demonstrated the potential biotechnological exploitation of the autotransporter secretion pathway, namely, by displaying heterologous proteins on the bacterial surface (453). The possible applications of this autodisplay system are numerous and include (i) exposure of antigenic determinants for vaccine development (142,267,316), (ii) expression of peptide libraries for epitope mapping or antibody specificity test (258), (iii) display of receptor or ligand for binding assays or purification (499,500), (iv) functional domain analyses of a heterologous protein (59,499,500), and (v) bioconversion by expressing enzymatic activity on the bacterial surface (229,230,276). In conclusion, further understanding of the autotransporter secretion pathway and the functions of the passenger domains will facilitate a richer view of the mechanisms and evolution of bacterial pathogenesis and provide important practical applications for the medical and biotechnological communities.…”

Fimbrial surface display systems in bacteria: from vaccines to random libraries

“…n, 1997). Recently, very promising results have also been obtained with systems based on autotransporter proteins, which are characterized by the fact that all information required to reach the bacterial surface resides in one single peptide chain (Maurer et al, 1997 ;Suzuki et al, 1995). Many of these systems have proven versatile with respect to the size and composition of the passenger sequences that can be displayed.…”