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-cha in (sc) Fab' by Bacillus brevis have been constructed. For the product ion of Fab', the cDNAs encoding the L chain and Fd' fragment (Fd with the hinge region) of a mouse-human chimeric Fab' against human urokina se-type plasminogen activator were fused directly with the translation -start and signal-peptide-encoding regions of the mwp gene, the gene f or one of the major cell-wall proteins of Bacillus brevis. The two fus ed genes were placed tandemly downstream from the promoter of the cell -wall protein gene operon (cwp) of B. brevis. For the production of sc Fab', the two cDNAs were linked with a synthetic oligonucleotide encod ing a flexible peptide linker of 17 or 24 amino acids, and fused with the translation start and signal-peptide-encoding regions of the mwp g ene. 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 cult ure, which is the highest level obtained so far for a gram-positive ba cterium. On the other hand, the scFab' remained at a level of a few mi lligrams per liter in the culture medium. The Fab' produced by B. brev is 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 fra gments.