Single-cell Characterization of Autotransporter-mediated Escherichia coli Surface Display of Disulfide Bond-containing Proteins

• Published in: The Journal of biological chemistry 2012-11, Vol.287 (46), p.38580-38589
• Main Authors: Ramesh, Balakrishnan, Sendra, Victor G, Cirino, Patrick C, Varadarajan, Navin
• Format: Article
• Language: English
• Subjects: Animals; Autotransporter; Biological Transport; Cell Membrane - metabolism; Cell Surface; Chymotrypsin; Chymotrypsin - chemistry; Cloning, Molecular; Disulfide; Disulfides - chemistry; Escherichia coli; Escherichia coli - metabolism; Flow Cytometry; Flow Cytometry - methods; Kinetics; Microbiology; Peptides - chemistry; Plasmids - metabolism; Protein Engineering - methods; Protein Structure, Secondary; Protein Transport; Rats; Recombinant Proteins - chemistry; Serine Protease; Serine Proteases - chemistry; Surface Properties
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M112.388199

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. Background: The ability of autotransporter (AT) to translocate polypeptides with multiple disulfide bonds is controversial. Results: Surface display of functional chymotrypsin (4 S-S) and M18 scFv (2 S-S) was quantitatively characterized. Conclusion: Surface display of functional recombinant protein with multiple disulfide bonds can be achieved using AT system. Significance: Displaying recombinant proteins with disulfide bonds enhances utility of ATs.