title: Cell-free synthesis of functional and endotoxin-free antibody Fab fragments by translocation into microsomes.
abstract: A eukaryotic cell-free system based on Spodoptera frugiperda cells was developed for the convenient synthesis of Fab antibody fragments and other disulfide bridge containing proteins. The system uses (i) a cell lysate that is mildly prepared under slightly reduced conditions, thus maintaining the activity of vesicles derived from the endoplasmic reticulum, (ii) signal peptide dependent translocation into these vesicles, and (iii) a redox potential based on reduced and oxidized glutathione. Monomeric heavy and light immunoglobulin chains are almost completely converted to highly active dimeric Fab joined by intermolecular disulfide bridges without supplementation of chaperones or protein disulfide isomerase. The applicability of the system is demonstrated by the synthesis of anti-lysozyme and anti-CD4 Fab antibody fragments yielding approximately 10 µg Fab per milliliter reaction mixture. The lack of endotoxins in this system is a prerequisite that synthesized Fab can be applied directly using whole synthesis reactions in cell-based assays that are sensitive to this substance class. Moreover, the system is compatible with PCR-generated linear templates enabling automated generation of antibody fragments in a high-throughput manner, and facilitating its application for screening and validation purposes.
authors.affiliation: RiNA Netzwerk RNA-Technologien GmbH, Berlin, Germany. Merk@RiNA-GmbH.eu
authors.forename: Helmut
authors.initials: H
authors.lastname: Merk
authors.forename: Christine
authors.initials: C
authors.lastname: Gless
authors.forename: Barbara
authors.initials: B
authors.lastname: Maertens
authors.forename: Michael
authors.initials: M
authors.lastname: Gerrits
authors.forename: Wolfgang
authors.initials: W
authors.lastname: Stiege
mesh_terms: D000818:Animals; D002474:Cell-Free System / Q000378:metabolism*; D004220:Disulfides; D004591:Electrophoresis, Polyacrylamide Gel; D004731:Endotoxins / Q000032:analysis; D057075:Enzyme Assays; D004921:Erythropoietin / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D006801:Humans; D007140:Immunoglobulin Fab Fragments / Q000134:cerebrospinal fluid* / Q000737:chemistry / Q000235:genetics; D008861:Microsomes / Q000378:metabolism*; D009113:Muramidase / Q000037:antagonists & inhibitors / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D018411:Spodoptera / Q000166:cytology* / Q000378:metabolism
chemical_list: D004220:Disulfides; D004731:Endotoxins; D007140:Immunoglobulin Fab Fragments; D011993:Recombinant Fusion Proteins; D004921:Erythropoietin; C421864:hen egg lysozyme; D009113:Muramidase