title: High-level production and secretion of a mouse-human chimeric Fab fragment with specificity to human carcino embryonic antigen in Escherichia coli.
abstract: A high-level secretion system for the production of mouse-human chimeric antibody 21B2 (MHC 21B2) Fab fragment specific for human carcino embryonic antigen (hCEA) in Escherichia coli has been constructed. The genes encoding a light chain and an Fd fragment (a variable region and the CH1 domain of a heavy chain) of a mouse-human chimeric antibody were directly fused to the signal peptide of the E. coli ompF gene sequence. E. coli cells containing expression vectors in which each of the two genes are located downstream of a separate tac promoter were able to secrete the light chain and Fd fragment as two of their major cellular proteins. The signal peptides were efficiently removed from the primary products by post-translational processing, although they formed insoluble aggregates, possibly in the periplasm. In high-cell-density culture experiments using a jar fermentor, the amount of light chain and Fd fragment produced was at levels of up to 2.88 g/l and 1.28 g/l culture, respectively. By optimizing the conditions that encourage correct folding, formation of disulphide bonds, and association of the light chain with the Fd fragment, we have established a procedure that can purify, re-fold, and combine aggregated products to electrophoretically homogeneous Fab fragment with a yield of approximately 47%. Fab fragment produced in this manner shows essentially the same antigen-binding activity and specificity to hCEA as the parental mouse antibody 21B2 (MoAb 21B2).
authors.affiliation: Biosciences Laboratory, Mitsubishi Kasei Corporation, Kanagawa, Japan.
authors.forename: T
authors.initials: T
authors.lastname: Shibui
authors.forename: K
authors.initials: K
authors.lastname: Munakata
authors.forename: R
authors.initials: R
authors.lastname: Matsumoto
authors.forename: K
authors.initials: K
authors.lastname: Ohta
authors.forename: R
authors.initials: R
authors.lastname: Matsushima
authors.forename: Y
authors.initials: Y
authors.lastname: Morimoto
authors.forename: K
authors.initials: K
authors.lastname: Nagahari
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000915:Antibody Affinity; D000918:Antibody Specificity; D001709:Biotechnology; D002272:Carcinoembryonic Antigen / Q000276:immunology*; D004926:Escherichia coli / Q000235:genetics*; D005803:Genes, Immunoglobulin; D005822:Genetic Vectors; D006801:Humans; D007140:Immunoglobulin Fab Fragments / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D051379:Mice; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification
chemical_list: D002272:Carcinoembryonic Antigen; D007140:Immunoglobulin Fab Fragments; D011994:Recombinant Proteins