Enrichment of Escherichia coli spheroplasts displaying scFv antibodies specific for antigens expressed on the human cell surface.
Anchored periplasmic expression (APEx) is a method for isolating high affinity ligand-binding proteins from large combinatorial libraries, and antibodies highly specific for soluble antigens were successfully isolated from APEx antibody libraries in combination with flow cytometric sorting (Harvey et al., Proc Natl Acad Sci USA 101(25):9193-9198, 2004). However, many disease markers and drug targets are localized on the cell surface, and often, unique posttranslational modifications and/or properly folded epitopes are lost when they were expressed and isolated in soluble form. In this study, we demonstrate that Escherichia coli spheroplasts, displaying antibodies and screened by a combination of plate-panning and flow cytometric sorting, can be used for isolating antibodies specific for antigens on the human cell surface. Two rounds of plate-panning followed by one round of flow cytometric sorting resulted in 7,200-fold enrichment of antibodies specific for the protective antigen of Bacillus anthracis from a large excess of spheroplasts expressing a scFv antibody to digoxin (a negative control). There is the potential to use this technique for library screening to find novel antibodies against disease cell surface antigens.
Qiu
Jun-Kang
JK
National Laboratory of Biomacromolecules, Center for Computational and Systems Biology, Institute of Biophysics, Chinese Academy of Sciences, No. 15 Datun Road, Chaoyang District, Beijing 100101, People's Republic of China.
Jung
Sang-Taek
ST
Georgiou
George
G
Hang
Hai-Ying
HY
D000942:Antigens, Bacterial / Q000276:immunology; D001427:Bacterial Toxins / Q000276:immunology; D004926:Escherichia coli / Q000235:genetics / Q000302:isolation & purification*; D005434:Flow Cytometry; D015870:Gene Expression; D006801:Humans; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D057127:Single-Chain Antibodies / Q000235:genetics / Q000378:metabolism*; D013104:Spheroplasts
D000942:Antigens, Bacterial; D001427:Bacterial Toxins; D011994:Recombinant Proteins; D057127:Single-Chain Antibodies; C030325:anthrax toxin