Bacterial Inner-membrane Display for Screening a Library of Antibody Fragments.
Antibodies engineered for intracellular function must not only have affinity for their target antigen, but must also be soluble and correctly folded in the cytoplasm. Commonly used methods for the display and screening of recombinant antibody libraries do not incorporate intracellular protein folding quality control, and, thus, the antigen-binding capability and cytoplasmic folding and solubility of antibodies engineered using these methods often must be engineered separately. Here, we describe a protocol to screen a recombinant library of single-chain variable fragment (scFv) antibodies for antigen-binding and proper cytoplasmic folding simultaneously. The method harnesses the intrinsic intracellular folding quality control mechanism of the Escherichia coli twin-arginine translocation (Tat) pathway to display an scFv library on the E. coli inner membrane. The Tat pathway ensures that only soluble, well-folded proteins are transported out of the cytoplasm and displayed on the inner membrane, thereby eliminating poorly folded scFvs prior to interrogation for antigen-binding. Following removal of the outer membrane, the scFvs displayed on the inner membrane are panned against a target antigen immobilized on magnetic beads to isolate scFvs that bind to the target antigen. An enzyme-linked immunosorbent assay (ELISA)-based secondary screen is used to identify the most promising scFvs for additional characterization. Antigen-binding and cytoplasmic solubility can be improved with subsequent rounds of mutagenesis and screening to engineer antibodies with high affinity and high cytoplasmic solubility for intracellular applications.
Fischell Department of Bioengineering, University of Maryland.
Parisa
P
Moghaddam-Taaheri
Department of Chemical and Biomolecular Engineering, University of Maryland.
Svetlana P
SP
Ikonomova
Department of Chemical and Biomolecular Engineering, University of Maryland.
Zifan
Z
Gong
Fischell Department of Bioengineering, University of Maryland.
Janna Q
JQ
Wisniewski
Fischell Department of Bioengineering, University of Maryland; Department of Chemical and Biomolecular Engineering, University of Maryland; ajkarl@umd.edu.
Amy J
AJ
Karlsson
D000941:Antigens; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli; D015723:Gene Library; D007128:Immunoglobulin Fragments; D019151:Peptide Library; D057127:Single-Chain Antibodies; D000069294:Twin-Arginine-Translocation System
D000941:Antigens; D007128:Immunoglobulin Fragments; D019151:Peptide Library; D057127:Single-Chain Antibodies; D000069294:Twin-Arginine-Translocation System