Anchored periplasmic expression, a versatile technology for the isolation of high-affinity antibodies from Escherichia coli-expressed libraries

Abstract

Anchored periplasmic expression (APEx) is a technology for the isolation of ligand-binding proteins from combinatorial libraries anchored on the periplasmic face of the inner membrane of Escherichia coli. After disruption of the outer membrane by Tris-EDTA-lysozyme, the inner-membrane-anchored proteins readily bind fluorescently labeled ligands as large as 240 kDa. Fluorescently labeled cells are isolated by flow cytometry, and the DNA of isolated clones is rescued by PCR. By using two rounds of APEx, the affinity of a neutralizing antibody to the Bacillus anthracis protective antigen was improved >200-fold, exhibiting a final K(D) of 21 pM. This approach has several technical advantages compared with previous library screening technologies, including the unique ability to screen for ligand-binding proteins that bind endogenously expressed ligands fused to a short-lived GFP. Further, APEx is able to display proteins either as an N-terminal fusion to a six-residue sequence derived from the native E. coli lipoprotein NlpA, or as a C-terminal fusion to the phage gene three minor coat protein of M13. The latter fusions allow hybrid phage display/APEx strategies without the need for further subcloning.

Fig. 1.

A schematic diagram showing the principle of APEx for the FC-based isolation of high-affinity antibody fragments.

Fig. 2.

Examples of targets visualized by APEx. (A) Fluorescence distribution of ABLEC cells expressing PA-specific (14B7), scFv (white), and Dig-specific (Dig) scFv (green), and labeled with 200 nM BODIPY-conjugated fluorescent antigens. Histograms represent the mean fluorescence intensity (M) of 10,000 E. coli events. (B) Histograms of cells expressing 14B7 scFv (white) or Dig scFv (green) labeled with 200 nM of the 240-kDa Dig-phycoerythrin conjugate.

Fig. 3.

Analysis of anti-PA antibody fragments selected using APEx. (A) SPR analysis of anti-PA scAb binding to PA. (B) Table of affinity data acquired by SPR. (C) FC histograms depicting the mean fluorescence (FL) intensity (M) of E. coli expressing anti-PA scFv clones in pAPEx1 and labeled with 200 nM PA-BODIPY conjugate as compared with those expressing the anti-Meth scFv as a negative control.

Fig. 4.

N-vs. C-terminal anchoring strategy comparison. Data represent the mean fluorescence intensity (M) of 10,000 E. coli events. (A) Anti-Dig scfv, anti-PA M18 scFv, and anti-Meth scFv expressed as N-terminal fusions in the pAPEx1 vector in E. coli specifically label with 200 nM of their respective antigen. (B) C-terminal fusions of same scFv in pAK200 vector specifically labeled with 200 nM of their respective antigen.

Fig. 5.

Fluorescent cell labeling using endogenously expressed antigen–GFP fusions. Histograms depict 10,000 E. coli events expressing (i) GFP–peptide fusion alone (mean = 13), (ii) GFP–peptide coexpressed with Dig scFv (mean = 15), (iii) GFP without peptide fusion coexpressed with 7C2 antipeptide scFv (mean = 14), and (iv) GFP–peptide coexpressed with 7C2 antipeptide scFv (mean = 46).