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). Using 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.

Ubiquibodies, Synthetic E3 Ubiquitin Ligases Endowed with Unnatural Substrate Specificity for Targeted Protein Silencing

“…These results also confirm the potential of CHIP for customizable target degradation. Indeed, given the plethora of existing DBPs against known cellular targets and the availability of robust technologies for on-demand isolation of new DBPs that function inside cells (26,36,37), ubiquibodies are likely to become a powerful tool for reverse genetics. In addition to the wide array of endogenous proteins that can be targeted with newly designed ubiquibodies, the existing R4-uAb construct described here could be used in conjunction with ␤-gal protein trapping (38) to silence virtually any ␤-galtagged protein expressed from its endogenous loci in cultured cells and whole organisms.…”

Bacterial Inner-membrane Display for Screening a Library of Antibody Fragments

“…By overexpressing a fusion of an scFv to the ssTorA signal sequence (the signal sequence from the TorA protein, which is naturally transported by the Tat pathway 20 ), translocation is stalled, resulting in display of scFvs on the inner membrane 19 . After enzymatic disruption of the outer membrane, the displayed antibodies are made available for screening for antigenbinding activity.…”

“…For screening to be successful, the scFv library being screened must be expressed as a fusion to the ssTorA signal peptide. Without this sequence, antibodies will not be directed to the Tat pathway and thus will not be translocated to the periplasm 19 . Additionally, it is imperative that a C-terminal epitope tag is fused to the antibodies to allow detection of the displayed antibodies in the binding assays.…”

“…Inner-membrane display can successfully isolate scFv antibodies with high levels of affinity for a target protein and high levels of cytoplasmic solubility. Additionally, subsequent rounds of directed evolution using inner-membrane display improve antibody characteristics 19 . To demonstrate this, an error-prone PCR library based on scFv13, which has a low level of binding affinity for β-galactosidase, was panned against the target antigen β-galactosidase using the display and panning method described in the protocol.…”

“…To improve the efficiency of engineering antibodies that are well folded in the cytoplasm, we previously reported the success of MAD-TRAP (membrane-anchored display for Tat-based recognition of associating proteins), a method for screening an scFv antibody library using Escherichia coli inner-membrane display 19 . Bacterial inner-membrane display relies on the twin-arginine translocation (Tat) pathway for transporting displayed antibodies, in contrast to other common display methods that use the secretory pathway.…”