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

J Immunol Methods. 2013 Aug 30;394(1-2):10-21. doi: 10.1016/j.jim.2013.04.010. Epub 2013 Apr 23.

Abstract

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.

Keywords: 3,3′,5,5′-tetramethylbenzidine; BSA; Bacteria; CFU; Chaperones; Cytoplasm; EC(50); ELISA; Fab; Fc; Fd; HRP; IPTG; M.O.I.; OD; PBS; PCR; PEG; PPIase; PVDF; Phage display; RT; SD; SDS; SEM; SPR; ScFv; Shine–Dalgarno sequence; Standard Error of the Mean; Surface Plasmon Resonance; TMB; VH; VL; bovine serum albumin; colony-forming units; dissociation constant; enzyme-linked immunosorbent assay; fragment crystallizable; fragment of antibody consisting of CH1 and VH; fragment-antigen-binding; half maximal effective concentration; horseradish peroxidase; huINSR; human insulin receptor; isopropyl β-d-1-thiogalactopyranoside; kd; multiplicity of infection; optical density; peptidyl prolyl cis-trans isomerase; phosphate buffer saline; polyethylene glycol; polymerase chain reaction; polyvinylidene fluoride; room temperature; single-chain variable fragment; sodium dodecyl sulfate; variable heavy; variable light.

MeSH terms

  • Enzyme-Linked Immunosorbent Assay
  • Escherichia coli / immunology*
  • Escherichia coli Proteins / physiology*
  • Immunoglobulin Fab Fragments / metabolism*
  • Membrane Proteins / physiology*
  • Peptide Library
  • Peptidylprolyl Isomerase / physiology*
  • Periplasm / metabolism*
  • Protein Folding

Substances

  • Escherichia coli Proteins
  • Immunoglobulin Fab Fragments
  • Membrane Proteins
  • Peptide Library
  • Peptidylprolyl Isomerase
  • FkpA protein, E coli