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

https://doi.org/10.1016/j.jim.2013.04.010Get rights and content
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Highlights

  • 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.

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.

Abbreviations

Fab
fragment-antigen-binding
Fc
fragment crystallizable
Fd
fragment of antibody consisting of CH1 and VH
ScFv
single-chain variable fragment
VH
variable heavy
VL
variable light
PPIase
peptidyl prolyl cis-trans isomerase
PCR
polymerase chain reaction
IPTG
isopropyl β-d-1-thiogalactopyranoside
OD
optical density
PBS
phosphate buffer saline
M.O.I.
multiplicity of infection
PEG
polyethylene glycol
HRP
horseradish peroxidase
ELISA
enzyme-linked immunosorbent assay
TMB
3,3′,5,5′-tetramethylbenzidine
huINSR
human insulin receptor
BSA
bovine serum albumin
kd
dissociation constant
RT
room temperature
EC50
half maximal effective concentration
CFU
colony-forming units
SD
Shine–Dalgarno sequence
PVDF
polyvinylidene fluoride
SPR
Surface Plasmon Resonance
SDS
sodium dodecyl sulfate
SEM
Standard Error of the Mean

Keywords

Chaperones
Bacteria
Cytoplasm
Phage display

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