Anchored periplasmic expression, a versatile technology for the isolation of high-affinity antibodies from Escherichia coli-expressed libraries.
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.
Harvey
Barrett R
BR
Institute for Cellular and Molecular Biology, University of Texas, Austin, 78712, USA.
Georgiou
George
G
Hayhurst
Andrew
A
Jeong
Ki Jun
KJ
Iverson
Brent L
BL
Rogers
Geoffrey K
GK
D000907:Antibodies, Bacterial / Q000032:analysis / Q000235:genetics*; D000942:Antigens, Bacterial / Q000032:analysis; D001408:Bacillus anthracis / Q000276:immunology; D001483:Base Sequence; D017931:DNA Primers; D004926:Escherichia coli / Q000235:genetics / Q000276:immunology*; D005434:Flow Cytometry; D015964:Gene Expression Regulation, Bacterial; D015723:Gene Library; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins / Q000032:analysis; D019897:Periplasm / Q000276:immunology; D016133:Polymerase Chain Reaction / Q000379:methods; D011993:Recombinant Fusion Proteins / Q000032:analysis
D000907:Antibodies, Bacterial; D000942:Antigens, Bacterial; D017931:DNA Primers; D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins