Efficient production of a functional mouse/human chimeric Fab' against human urokinase-type plasminogen activator by Bacillus brevis.
Expression/secretion vectors for the production of Fab' and single-chain (sc) Fab' by Bacillus brevis have been constructed. For the production of Fab', the cDNAs encoding the L chain and Fd' fragment (Fd with the hinge region) of a mouse-human chimeric Fab' against human urokinase-type plasminogen activator were fused directly with the translation-start and signal-peptide-encoding regions of the mwp gene, the gene for one of the major cell-wall proteins of Bacillus brevis. The two fused genes were placed tandemly downstream from the promoter of the cell-wall protein gene operon (cwp) of B. brevis. For the production of scFab', the two cDNAs were linked with a synthetic oligonucleotide encoding a flexible peptide linker of 17 or 24 amino acids, and fused with the translation start and signal-peptide-encoding regions of the mwp gene. Fab' was efficiently produced by B. brevis, being accumulated at a level of 100 mg/l in the culture medium in a simple shake-flask culture, which is the highest level obtained so far for a gram-positive bacterium. On the other hand, the scFab' remained at a level of a few milligrams per liter in the culture medium. The Fab' produced by B. brevis showed comparable antigen-binding activity to that of the parental antibody. The L chain and Fd' fragment, constituting the Fab', had the correct N-terminal amino acid sequences. These results indicate that B. brevis is a very promising host for the production of native Ig fragments.
Inoue
Y
Y
Department of Applied Biological Sciences, Faculty of Agriculture, Nagoya University, Japan.
Ohta
T
T
Tada
H
H
Iwasa
S
S
Udaka
S
S
Yamagata
H
H
D000595:Amino Acid Sequence; D000818:Animals; D000918:Antibody Specificity; D001407:Bacillus / Q000378:metabolism*; D001483:Base Sequence; D005798:Genes, Bacterial; D005813:Genes, Synthetic; D006801:Humans; D007140:Immunoglobulin Fab Fragments / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology* / Q000302:isolation & purification; D007074:Immunoglobulin G / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology* / Q000302:isolation & purification; D051379:Mice; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000276:immunology* / Q000302:isolation & purification; D014568:Urokinase-Type Plasminogen Activator / Q000276:immunology*
D007140:Immunoglobulin Fab Fragments; D007074:Immunoglobulin G; D011993:Recombinant Fusion Proteins; D014568:Urokinase-Type Plasminogen Activator