title: Engineering antibody fitness and function using membrane-anchored display of correctly folded proteins.
abstract: A hallmark of the bacterial twin-arginine translocation (Tat) pathway is its ability to export folded proteins. Here, we discovered that overexpressed Tat substrate proteins form two distinct, long-lived translocation intermediates that are readily detected by immunolabeling methods. Formation of the early translocation intermediate Ti-1, which exposes the N- and C-termini to the cytoplasm, did not require an intact Tat translocase, a functional Tat signal peptide, or a correctly folded substrate. In contrast, formation of the later translocation intermediate, Ti-2, which exhibits a bitopic topology with the N-terminus in the cytoplasm and C-terminus in the periplasm, was much more particular, requiring an intact translocase, a functional signal peptide, and a correctly folded substrate protein. The ability to directly detect Ti-2 intermediates was subsequently exploited for a new protein engineering technology called MAD-TRAP (membrane-anchored display for Tat-based recognition of associating proteins). Through the use of just two rounds of mutagenesis and screening with MAD-TRAP, the intracellular folding and antigen-binding activity of a human single-chain antibody fragment were simultaneously improved. This approach has several advantages for library screening, including the unique involvement of the Tat folding quality control mechanism that ensures only native-like proteins are displayed, thus eliminating poorly folded sequences from the screening process.
authors.affiliation: School of Chemical and Biomolecular Engineering, Cornell University, Ithaca, NY 14853, USA.
authors.forename: Amy J
authors.initials: AJ
authors.lastname: Karlsson
authors.forename: Hyung-Kwon
authors.initials: HK
authors.lastname: Lim
authors.forename: Hansen
authors.initials: H
authors.lastname: Xu
authors.forename: Mark A
authors.initials: MA
authors.lastname: Rocco
authors.forename: Matthew A
authors.initials: MA
authors.lastname: Bratkowski
authors.forename: Ailong
authors.initials: A
authors.lastname: Ke
authors.forename: Matthew P
authors.initials: MP
authors.lastname: DeLisa
mesh_terms: D000941:Antigens / Q000276:immunology; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism; D003593:Cytoplasm / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000235:genetics* / Q000378:metabolism*; D007150:Immunohistochemistry / Q000379:methods; D008024:Ligands; D026901:Membrane Transport Proteins / Q000235:genetics* / Q000378:metabolism*; D016296:Mutagenesis / Q000235:genetics; D019897:Periplasm / Q000235:genetics / Q000378:metabolism; D015202:Protein Engineering / Q000379:methods*; D017510:Protein Folding; D021382:Protein Sorting Signals / Q000235:genetics; D021381:Protein Transport; D057127:Single-Chain Antibodies / Q000235:genetics / Q000378:metabolism
chemical_list: D000941:Antigens; D002352:Carrier Proteins; D029968:Escherichia coli Proteins; D008024:Ligands; D026901:Membrane Transport Proteins; D021382:Protein Sorting Signals; D057127:Single-Chain Antibodies; C408585:twin-arginine translocase complex, E coli