Isolation of full-length IgG antibodies from combinatorial libraries expressed in Escherichia coli.
We have developed a technology for the facile isolation of full-length IgG antibodies with desired specificity from combinatorial libraries expressed in Escherichia coli. Full-length heavy and light chains are expressed from a bicistronic operon and are secreted into the periplasm where they assemble into aglycosylated IgGs that are fully functional for antigen binding. Expression of an inner membrane-tethered Fc-binding protein is used to capture the IgG molecules and anchor them to the cell. Following outer membrane disruption, clones expressing IgGs that specifically recognize fluorescently labeled antigen are selected by flow cytometry. This technique was used for the isolation of several IgGs with nanomolar affinities toward the protective antigen of Bacillus anthracis from immune libraries. High-throughput isolation of E. coli-derived full-length IgG can greatly expedite the discovery and production of antibodies for therapeutic and diagnostic applications.
Mazor
Yariv
Y
University of Texas, Austin, TX, USA.
Van Blarcom
Thomas
T
Iverson
Brent L
BL
Georgiou
George
G
D000818:Animals; D002999:Clone Cells; D003001:Cloning, Molecular; D020650:Combinatorial Chemistry Techniques / Q000379:methods*; D018076:DNA, Complementary / Q000096:biosynthesis; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli / Q000378:metabolism*; D005434:Flow Cytometry; D006801:Humans; D007074:Immunoglobulin G / Q000302:isolation & purification*; D007143:Immunoglobulin Heavy Chains / Q000235:genetics; D007147:Immunoglobulin Light Chains / Q000235:genetics; D007135:Immunoglobulin Variable Region / Q000235:genetics; D051379:Mice; D019151:Peptide Library; D019897:Periplasm / Q000378:metabolism; D010957:Plasmids / Q000235:genetics; D016133:Polymerase Chain Reaction; D012313:RNA / Q000302:isolation & purification; D017422:Sequence Analysis, DNA; D013104:Spheroplasts / Q000378:metabolism; D013154:Spleen / Q000378:metabolism
D018076:DNA, Complementary; D007074:Immunoglobulin G; D007143:Immunoglobulin Heavy Chains; D007147:Immunoglobulin Light Chains; D007135:Immunoglobulin Variable Region; D019151:Peptide Library; D012313:RNA