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APEx 2-hybrid, a quantitative protein-protein interaction assay for antibody discovery and engineering - PubMed

  • ️Mon Jan 01 2007

APEx 2-hybrid, a quantitative protein-protein interaction assay for antibody discovery and engineering

Ki Jun Jeong et al. Proc Natl Acad Sci U S A. 2007.

Abstract

We have developed a bacterial system for the discovery of interacting proteins that, unlike other two-hybrid technologies, allows for the selection of protein pairs on the basis of affinity or expression. This technology relies on the anchored periplasmic expression (APEx) of one protein (bait) on the periplasmic side of the inner membrane of Escherichia coli and its interacting partner (prey) as a soluble, epitope-tagged, periplasmic protein. Upon removal of the outer membrane by spheroplasting, periplasmic proteins, including any unbound epitope-tagged prey, are released into the extracellular fluid. However, if the epitope-tagged prey can bind to the membrane-anchored bait, it remains associated with the cell and can be detected quantitatively by using fluorescent anti-epitope tag antibodies. Cells expressing prey:bait pairs exhibiting different affinities can be readily distinguished by flow cytometry. The utility of this technology, called APEx two-hybrid, was demonstrated in two demanding antibody engineering applications: First, single-chain variable fragment (scFvs) with increased affinity to the protective antigen of Bacillus anthracis were isolated from cells coexpressing libraries of scFv random mutants, together with endogenously expressed antigen. Second, APEx two-hybrid coupled with multicolor FACS analysis to account for protein expression was used for the selection of mutant Fab antibody fragments exhibiting improved expression in the bacterial periplasm.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.

Flow-cytometric schemes and assays by APEx two-hybrid system. (A) A schematic diagram showing the principle of the APEx two-hybrid system. (B) Fluorescence distribution of E. coli cells coexpressing NlpA-scFvs and PelB-[PA-D4wt-FLAG] or PA-D4 mutants (Y681A or Y688A). Spheroplasted cells were labeled with anti-FLAG-FITC conjugates. Mn represents the mean fluorescence intensity of the spheroplast population as determined by forward scatter (FSC) vs. side scatter (SSC).

Fig. 2.
Fig. 2.

Analysis of anti-PA antibody fragments selected by APEx two-hybrid. (A) Flow cytometry histogram depicting the mean fluorescence (Mn) of E. coli expressing anti-PA scFvs and labeled with anti-FLAG-FITC. (B) BIACore analysis of anti-PA scAb binding to PA. (C) Rate constants for antigen binding and dissociation acquired by surface plasmon resonance (SPR).

Fig. 3.
Fig. 3.

Analysis of Fab assembly and expression of APEx two-hybrid system. (A) A schematic diagram of Fab assembly and expression analysis by APEx two-hybrid. The light chain is coexpressed with heavy chain fused to a FLAG tag. Antibody assembly occurs in the periplasm and, after spheroplasting, the cells are labeled with anti-FLAG-PE and PA-FITC, which were used to detect Fab expression and antigen binding, respectively. (B and C) Cells expressing M18.1 hum Fab were grown at different temperatures [37°C (white) and 25°C (red)] and labeled with anti-FLAG-PE (B) or PA-FITC (C), which represent expression and antigen binding, respectively. (D) Fab was expressed in soluble form in cells grown at both temperatures, and the yields of antigen binding Fabs were analyzed by ELISA.

Fig. 4.
Fig. 4.

Expression maturation of Fab antibodies by APEx two-hybrid. (A and B) Cells expressing Fab no. 3 or Fab no. 28 isolated by FACS were grown at 37°C, and labeled with anti-FLAG-FITC (for expression) (A) and PA-FITC (for activity) (B). (C) The yields of M18.1 hum Fab, clone no. 3, and no. 28 were determined by ELISA.

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