[Anchored periplasmic expression, a versatile technology for the isolation of high-affinity antibodies from Escherichia coli-expressed libraries]
>>Published online before print June 14, 2004
Proc. Natl. Acad. Sci. USA, 10.1073/pnas.0400187101
Applied Biological Sciences
Anchored periplasmic expression, a versatile technology for the isolation of high-affinity antibodies from Escherichia coli-expressed libraries
Barrett R. Harvey *, George Georgiou *, Andrew Hayhurst *, Ki Jun Jeong *, Brent L. Iverson *¶, and Geoffrey K. Rogers
*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, and approved May 10, 2004 (received for review January 9, 2004)
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 KD 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.<<
Cheers, Tuck |
