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

doi:10.1073/pnas.0400187101

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

^*Institute for Cellular and Molecular Biology and Departments of ^†Chemical Engineering, ^¶Chemistry and Biochemistry, and ^‡Biomedical Engineering, University of Texas, Austin, TX 78712

Edited by James A. Wells, Sunesis Pharmaceuticals, Inc., South San Francisco, CA (received for review January 9, 2004)

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.

Footnotes

↵ § To whom correspondence may be addressed. E-mail: gg{at}che.utexas.edu or biverson{at}mail.utexas.edu.
This paper was submitted directly (Track II) to the PNAS office.
Abbreviations: APEx, anchored periplasmic expression; FC, flow cytometry; g3p, M13 phage gene 3 minor coat protein; PA, protective antigen; Dig, digoxigenin; SPR, surface plasmon resonance; PI, propidium iodide; Meth, methamphetamine; BODIPY, 4,4-difluoro-4-bora-3{α},4{α}-diaza-s-indacene; scFv, single-chain variable fragment; scAb, single-chain Ab fragment.