1. Efficient production of a functional mouse/human chimeric Fab′ against human urokinase-type plasminogen activator by Bacillus brevis
- Author
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Susumu Iwasa, Hideo Yamagata, Shigezo Udaka, Hiroko Tada, Toshihiro Ohta, and Y. Inoue
- Subjects
Operon ,Recombinant Fusion Proteins ,Molecular Sequence Data ,Bacillus ,Applied Microbiology and Biotechnology ,Immunoglobulin Fab Fragments ,Mice ,Antibody Specificity ,Complementary DNA ,Gene expression ,Genes, Synthetic ,Animals ,Humans ,Amino Acid Sequence ,Promoter Regions, Genetic ,Gene ,Peptide sequence ,chemistry.chemical_classification ,Base Sequence ,biology ,General Medicine ,biology.organism_classification ,Urokinase-Type Plasminogen Activator ,Molecular biology ,Amino acid ,Biochemistry ,chemistry ,Brevibacillus brevis ,Genes, Bacterial ,Immunoglobulin G ,Biotechnology - Abstract
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.
- Published
- 1997