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

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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 KID 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.
Publisher
NATL ACAD SCIENCES
Issue Date
2004-06
Language
English
Article Type
Article
Keywords

PHAGE-DISPLAY; SURFACE DISPLAY; FRAGMENTS; PROTEIN; MEMBRANE; CELL; GENE; BACTERIA; SYSTEM; LIPOPROTEIN

Citation

PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, v.101, no.25, pp.9193 - 9198

ISSN
0027-8424
DOI
10.1073/pnas.0400187101
URI
http://hdl.handle.net/10203/83173
Appears in Collection
CBE-Journal Papers(저널논문)
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