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

Y. Inoue, T. Ohta, Hiroko Tada, S. Iwasa, S. Udaka, H. Yamagata

Research output: Contribution to journalArticle

18 Citations (Scopus)

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.

Original languageEnglish
Pages (from-to)487-492
Number of pages6
JournalApplied Microbiology and Biotechnology
Volume48
Issue number4
DOIs
Publication statusPublished - 1997

Fingerprint

Urokinase-Type Plasminogen Activator
Bacilli
Bacillus
Genes
Protein Sorting Signals
Cell Wall
Culture Media
Complementary DNA
Amino acids
Amino Acids
Batch Cell Culture Techniques
Gram-Positive Bacteria
Proteins
Hinges
Operon
Oligonucleotides
Amino Acid Sequence
Antigens
Bacteria
Antibodies

ASJC Scopus subject areas

  • Biotechnology
  • Microbiology
  • Bioengineering

Cite this

Efficient production of a functional mouse/human chimeric Fab' against human urokinase-type plasminogen activator by Bacillus brevis. / Inoue, Y.; Ohta, T.; Tada, Hiroko; Iwasa, S.; Udaka, S.; Yamagata, H.

In: Applied Microbiology and Biotechnology, Vol. 48, No. 4, 1997, p. 487-492.

Research output: Contribution to journalArticle

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