Secretion of a functional Fab fragment in Escherichia coli and the influence of culture conditions.
Genes encoding a light chain and an Fd region (a variable region and a CH1 domain of a heavy chain) of a mouse-human chimeric antibody with specificity for human carcinoembryonic antigen (CEA) were fused to a DNA segment coding for the signal peptide of Escherichia coli ompF. E. coli cells harbouring an expression vector containing these genes downstream of a tac promoter were able to secrete a Fab fragment of the antibody efficiently. When the cells were cultured at 37 degrees C and the inducer (isopropyl-beta-D-thiogalactopyranoside, IPTG) concentration was 1 mM (standard conditions), production of functional Fab was very low (medium; 200 ng/l culture and periplasm; less than 90 ng/l culture). In order to optimize functional Fab production, we examined the influence of culture conditions (i.e. temperature and the inducer concentration) on secretion of the product. It was found that a 12.7-fold higher amount of Fab fragment could be produced at 30 degrees C using 0.1 mM IPTG, as compared with standard conditions. Under these optimal conditions, functional Fab accumulated in the periplasm and culture medium for 10 h after induction and the total production level was found to reach approximately 4.5 mg/l culture.
Biosciences Laboratory, Mitsubishi Kasei Corporation, Kanagawa, Japan.
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Shibui
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Nagahari
D000818:Animals; D001483:Base Sequence; D001709:Biotechnology; D002272:Carcinoembryonic Antigen; D003470:Culture Media; D004247:DNA / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics*; D005803:Genes, Immunoglobulin; D006801:Humans; D007140:Immunoglobulin Fab Fragments / Q000235:genetics*; D051379:Mice; D008969:Molecular Sequence Data; D010957:Plasmids; D011994:Recombinant Proteins / Q000235:genetics
D002272:Carcinoembryonic Antigen; D003470:Culture Media; D007140:Immunoglobulin Fab Fragments; D011994:Recombinant Proteins; D004247:DNA