title: Production of functional antibody fragments in a vesicle-based eukaryotic cell-free translation system.
abstract: Cell-free protein synthesis is of increasing interest for the rapid and high-throughput synthesis of many proteins, in particular also antibody fragments. In this study, we present a novel strategy for the production of single chain antibody fragments (scFv) in a eukaryotic in vitro translation system. This strategy comprises the cell-free expression, isolation and label-free interaction analysis of a model antibody fragment synthesized in two differently prepared insect cell lysates. These lysates contain translocationally active microsomal structures derived from the endoplasmic reticulum (ER), allowing for posttranslational modifications of cell-free synthesized proteins. Both types of these insect cell lysates enable the synthesis and translocation of scFv into ER-derived vesicles. However, only the one that has a specifically adapted redox potential yields functional active antibody fragments. We have developed a new methodology for the isolation of functional target proteins based on the translocation of cell-free produced scFv into microsomal structures and subsequent collection of protein-enriched vesicles. Antibody fragments that have been released from these vesicles are shown to be well suited for label-free binding studies. Altogether, these results show the potential of insect cell lysates for the production, purification and selection of antibody fragments in an easy-to-handle and time-saving manner.
authors.affiliation: Fraunhofer Institute for Biomedical Engineering, Branch Potsdam-Golm, Potsdam, Germany.
authors.forename: Marlitt
authors.initials: M
authors.lastname: Stech
authors.forename: Helmut
authors.initials: H
authors.lastname: Merk
authors.forename: Jörg A
authors.initials: JA
authors.lastname: Schenk
authors.forename: Walter F M
authors.initials: WF
authors.lastname: Stöcklein
authors.forename: Doreen A
authors.initials: DA
authors.lastname: Wüstenhagen
authors.forename: Burkhard
authors.initials: B
authors.lastname: Micheel
authors.forename: Claus
authors.initials: C
authors.lastname: Duschl
authors.forename: Frank F
authors.initials: FF
authors.lastname: Bier
authors.forename: Stefan
authors.initials: S
authors.lastname: Kubick
mesh_terms: D000818:Animals; D019149:Bioreactors; D001709:Biotechnology; D018929:Cell Culture Techniques / Q000379:methods*; D002474:Cell-Free System / Q000378:metabolism*; D017077:Culture Media, Conditioned; D008555:Melitten / Q000235:genetics; D010084:Oxidation-Reduction; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D061987:Sf9 Cells; D057127:Single-Chain Antibodies / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D014162:Transfection
chemical_list: D017077:Culture Media, Conditioned; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; D057127:Single-Chain Antibodies; D008555:Melitten