Applied Microbiology and Biotechnology

, Volume 48, Issue 4, pp 487–492 | Cite as

Efficient production of a functional mouse/human chimeric Fab′ against human urokinase-type plasminogen activator by Bacillus brevis

  • Y. Inoue
  • T. Ohta
  • H. Tada
  • S. Iwasa
  • S. Udaka
  • H. Yamagata
ORIGINAL PAPER

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.

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Copyright information

© Springer-Verlag Berlin Heidelberg 1997

Authors and Affiliations

  • Y. Inoue
    • 2
  • T. Ohta
    • 1
  • H. Tada
    • 4
  • S. Iwasa
    • 3
  • S. Udaka
    • 5
  • H. Yamagata
    • 1
  1. 1.School of Life Science, Tokyo University of Pharmacy and Life Science, 1432-1, Horinouchi, Hachioji, Tokyo 192-03, Japan Tel.: +81 426 76 7053 Fax: +81 426 76 7081 e-mail: yamagata@ls.toyaku.ac.jpJP
  2. 2.Department of Applied Biological Sciences, Faculty of Agriculture, Nagoya University, Chikusa, Nagoya 464, JapanJP
  3. 3.DDS Research Laboratories, Takeda Chemical Industries Ltd., 2-17-85 Jusohonmachi, Yodogawa-ku, Osaka 532, JapanJP
  4. 4.Biotechnology Research Laboratories, Takeda Chemical Industries Ltd., Osaka 532, JapanJP
  5. 5.Department of Brewing and Fermentation, Faculty of Agriculture, Tokyo University of Agriculture, 1-1-1 Sakuragaoka, Setagaya-ku, Tokyo 156, JapanJP

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