title: Bacterial cytoplasmic display platform Retained Display (ReD) identifies stable human germline antibody frameworks.
abstract: Conventional antibody surface display requires fusion protein export through at least one cellular membrane, constraining the yield and occasioning difficulties in achieving scaled production. To circumvent this limitation, we developed a novel cytoplasmic display platform, Retained Display (ReD), and used it to screen for human scFv frameworks that are highly soluble and stable in the bacterial cytoplasm. ReD, based on the retention of high-molecular weight complexes within detergent-permeabilized Escherichia coli, enabled presentation of exogenous targets to antibodies that were expressed and folded in the cytoplasm. All human λ and κ light chain family genes were expressed as IGHV3-23 fusions. Members of the λ subfamilies 1, 3 and 6 were soluble cytoplasmic partners of IGHV3-23. Contrary to previous in vivo screens for soluble reduced scFvs, the pairings identified by ReD were identical to the human germline sequences for the framework, CDR1 and CDR2 regions. Using the most soluble scFv scaffold identified, we demonstrated tolerance to CDR3 diversification and isolated a binding scFv to an exogenous protein target. This screening system has the potential to rapidly produce antibodies to target threats such as emerging infectious diseases and bioterror agents.
authors.affiliation: affinity BIO Pty Ltd., Melbourne, VIC, Australia.
authors.forename: Matthew D
authors.initials: MD
authors.lastname: Beasley
authors.forename: Keith P
authors.initials: KP
authors.lastname: Niven
authors.forename: Wendy R
authors.initials: WR
authors.lastname: Winnall
authors.forename: Ben R
authors.initials: BR
authors.lastname: Kiefel
mesh_terms: D001324:Autoantigens / Q000235:genetics; D003001:Cloning, Molecular; D003593:Cytoplasm / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D009419:Nerve Tissue Proteins / Q000235:genetics; D057127:Single-Chain Antibodies / Q000096:biosynthesis* / Q000235:genetics
chemical_list: D001324:Autoantigens; C063762:CDR1 protein, human; C095184:CDR2 protein, human; D009419:Nerve Tissue Proteins; D057127:Single-Chain Antibodies
keywords: Bacterial display; Intrabodies; Phage display; Retained display; Single chain Fv