Enhancement of antibody fragment secretion into the Escherichia coli periplasm by co-expression with the peptidyl prolyl isomerase, FkpA, in the cytoplasm

• Published in: Journal of immunological methods 2013-08, Vol.394 (1-2), p.10-21
• Main Authors: Levy, Raphael, Ahluwalia, Kiran, Bohmann, David J., Giang, Hoa M., Schwimmer, Lauren J., Issafras, Hassan, Reddy, Nithin B., Chan, Chung, Horwitz, Arnold H., Takeuchi, Toshihiko
• Format: Article
• Language: English
• Subjects: antibodies; Bacteria; bacteriophages; Chaperones; clones; Cytoplasm; cytosol; DNA libraries; Enzyme-Linked Immunosorbent Assay; Escherichia coli; Escherichia coli - immunology; Escherichia coli Proteins - physiology; Immunoglobulin Fab Fragments - metabolism; Membrane Proteins - physiology; Peptide Library; Peptidylprolyl Isomerase - physiology; Periplasm - metabolism; Phage display; Protein Folding; screening; secretion; signal peptide
• ISSN: 0022-1759; 1872-7905
• DOI: 10.1016/j.jim.2013.04.010

Improper protein folding or aggregation can frequently be responsible for low expression and poor functional activity of antibody fragments secreted into the Escherichia coli periplasm. Expression issues also can affect selection of antibody candidates from phage libraries, since antibody fragments displayed on phage also are secreted into the E. coli periplasm. To improve secretion of properly folded antibody fragments into the periplasm, we have developed a novel approach that involves co-expressing the antibody fragments with the peptidyl prolyl cis-trans isomerase, FkpA, lacking its signal sequence (cytFkpA) which consequently is expressed in the E. coli cytosol. Cytoplasmic expression of cytFkpA improved secretion of functional Fab fragments into the periplasm, exceeding even the benefits from co-expressing Fab fragments with native, FkpA localized in the periplasm. In addition, panning and subsequent screening of large Fab and scFv naïve phage libraries in the presence of cytFkpA significantly increased the number of unique clones selected, as well as their functional expression levels and diversity. •Expression method raising periplasmic secretion of functional antibody fragments•Novel method reveals more unique clones from scFv or Fab naïve phage libraries.•We demonstrate elevated expression levels and diversity of selected phage clones.