Asunto(s)
Anticuerpos/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Periplasma/metabolismo , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/metabolismo , Secuencia de Aminoácidos , Anticuerpos/genética , Anticuerpos/inmunología , Anticuerpos/aislamiento & purificación , Secuencia de Bases , Escherichia coli/citología , Fermentación , Expresión Génica , Fragmentos de Péptidos/biosíntesis , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/inmunología , Fragmentos de Péptidos/metabolismo , Periplasma/genética , Plásmidos/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunologíaRESUMEN
We demonstrate the importance of optimizing the balance of light chain (LC) and heavy chain (HC) expression to achieve high level production of Fab' fragments in the Escherichia coli periplasm. The LC:HC balance has been controlled by varying the codon usage of the signal peptide (SP) and 5' mature domain coding regions. Different SP coding regions have been identified from a codon wobble-based library using alkaline phosphatase (AP) as a reporter gene. A plasmid system that enables random combination of these variant SP coding regions is used to construct optimized Fab' expression plasmids. These small plasmid libraries facilitated selection of optimal Fab' expression plasmids and resulted in increases of periplasmic yield, up to 580 mgL(-1) from E. coli fermentations and will enable rapid variable region subcloning and selection of future Fab(') expression plasmids.