USE OF MUTATOR CELLS AS A MEANS FOR INCREASING PRODUCTION LEVELS OF ARECOMBINANT ANTIBODY-DIRECTED AGAINST HEPATITIS-B

A mutation strategy which utilises phage display technology and the Es cherichia coli mutator strains, mutD5-FIT and XL1-RED, was applied to a Hepatitis B (HepB) specific single-chain Fv (scFv) to incorporate ra ndom mutations throughout the gene. Messenger RNA from a hybridoma pro ducing antibodies against HepB was isolated, reverse transcribed and u sed as template for the production of scFv. Following production of th e scFv protein using an E. coli expression vector (pGC), the scFv gene was recloned into a phage display vector (pHFA). This gene construct was introduced into E. coli mutator cells and the transformed cells we re used as an inoculum for liquid cultures. After five cycles of growt h at 37 degrees C, each followed by dilution and re-inoculation of fre sh media, recombinant phage were recovered. Nucleotide sequence analys is of the scFv gene in phage selected on HBsAg-coated magnetic beads i dentified amino acid substitutions which produced an increase of great er than 10-fold in apparent production levels. Competitive ELISA studi es showed that the selected scFv mutants appeared to have similar affi nity to HBsAg as the parent scFv. The apparent increase in production was not the result of improved surface characteristics of regions uniq uely exposed in scFvs, as the sites did not correlate with the variabl e/constant interface of the scFv variable region normally masked in Fa bs or IgGs. (C) 1997 Elsevier Science B.V.