title: Stable expression and purification of a functional processed Fab' fragment from a single nascent polypeptide in CHO cells expressing the mCAT-1 retroviral receptor.
abstract: Monoclonal antibodies and derivative formats such as Fab' fragments are used in a broad range of therapeutic, diagnostic and research applications. New systems and methodologies that can improve the production of these proteins are consequently of much interest. Here we present a novel approach for the rapid production of processed Fab' fragments in a CHO cell line that has been engineered to express the mouse cationic amino acid transporter receptor 1 (mCAT-1). This facilitated the introduction of the target antibody gene through retroviral transfection, rapidly producing stable expression. Using this system, we designed a single retroviral vector construct for the expression of a target Fab' fragment as a single polypeptide with a furin cleavage site and a FMDV 2A self-cleaving peptide introduced to bridge the light and truncated heavy chain regions. The introduction of these cleavage motifs ensured equimolar expression and processing of the heavy and light domains as exemplified by the production of an active chimeric Fab' fragment against the Fas receptor, routinely expressed in 1-2mg/L yield in spinner-flask cell cultures. These results demonstrate that this method could have application in the facile production of bioactive Fab' fragments.
authors.affiliation: Centre for Infection and Immunity, School of Medicine, Dentistry and Biomedical Science, Queen's University of Belfast, 97 Lisburn Rd, Belfast BT9 7BL, Northern Ireland, United Kingdom.
authors.forename: Nicolas
authors.initials: N
authors.lastname: Camper
authors.forename: Teresa
authors.initials: T
authors.lastname: Byrne
authors.forename: Roberta E
authors.initials: RE
authors.lastname: Burden
authors.forename: Jenny
authors.initials: J
authors.lastname: Lowry
authors.forename: Breena
authors.initials: B
authors.lastname: Gray
authors.forename: James A
authors.initials: JA
authors.lastname: Johnston
authors.forename: Marie E
authors.initials: ME
authors.lastname: Migaud
authors.forename: Shane A
authors.initials: SA
authors.lastname: Olwill
authors.forename: Richard J
authors.initials: RJ
authors.lastname: Buick
authors.forename: Christopher J
authors.initials: CJ
authors.lastname: Scott
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis* / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification; D015153:Blotting, Western; D016466:CHO Cells; D027201:Cationic Amino Acid Transporter 1 / Q000235:genetics / Q000276:immunology*; D006224:Cricetinae; D003412:Cricetulus; D004797:Enzyme-Linked Immunosorbent Assay; D005822:Genetic Vectors / Q000235:genetics; D007140:Immunoglobulin Fab Fragments / Q000096:biosynthesis* / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification; D012190:Retroviridae / Q000235:genetics; D014162:Transfection / Q000379:methods*; D019014:fas Receptor / Q000276:immunology
chemical_list: D000911:Antibodies, Monoclonal; D027201:Cationic Amino Acid Transporter 1; D007140:Immunoglobulin Fab Fragments; D019014:fas Receptor