APEx 2-hybrid, a quantitative protein-protein interaction assay for antibody discovery and engineering.
We have developed a bacterial system for the discovery of interacting proteins that, unlike other two-hybrid technologies, allows for the selection of protein pairs on the basis of affinity or expression. This technology relies on the anchored periplasmic expression (APEx) of one protein (bait) on the periplasmic side of the inner membrane of Escherichia coli and its interacting partner (prey) as a soluble, epitope-tagged, periplasmic protein. Upon removal of the outer membrane by spheroplasting, periplasmic proteins, including any unbound epitope-tagged prey, are released into the extracellular fluid. However, if the epitope-tagged prey can bind to the membrane-anchored bait, it remains associated with the cell and can be detected quantitatively by using fluorescent anti-epitope tag antibodies. Cells expressing prey:bait pairs exhibiting different affinities can be readily distinguished by flow cytometry. The utility of this technology, called APEx two-hybrid, was demonstrated in two demanding antibody engineering applications: First, single-chain variable fragment (scFvs) with increased affinity to the protective antigen of Bacillus anthracis were isolated from cells coexpressing libraries of scFv random mutants, together with endogenously expressed antigen. Second, APEx two-hybrid coupled with multicolor FACS analysis to account for protein expression was used for the selection of mutant Fab antibody fragments exhibiting improved expression in the bacterial periplasm.
Jeong
Ki Jun
KJ
Department of Chemical Engineering, Institute for Cellular and Molecular Biology, University of Texas, Austin, TX 78712, USA.
Seo
Min Jeong
MJ
Iverson
Brent L
BL
Georgiou
George
G
D000906:Antibodies / Q000378:metabolism*; D000915:Antibody Affinity / Q000276:immunology; D000941:Antigens / Q000378:metabolism; D005434:Flow Cytometry; D006801:Humans; D007135:Immunoglobulin Variable Region / Q000276:immunology; D019897:Periplasm / Q000378:metabolism*; D011485:Protein Binding; D015202:Protein Engineering; D020798:Two-Hybrid System Techniques
D000906:Antibodies; D000941:Antigens; D007135:Immunoglobulin Variable Region