DIFFERENCES IN SEQUENCE-SPECIFIC EXPRESSION OF 2 ANTI-ARSONATE FABS IN ESCHERICHIA-COLI

Monoclonal antibodies are potentially useful therapeutic agents and ca n now be produced in hosts such as bacteria. However, it has been foun d that bacterial expression of some antibody-combining site fragments is greatly diminished. We compared two homologous anti-arsonate antibo dies, 36-65 and 36-71, to address the question of why the former but n ot the latter expresses well as Fab in E. coli. These antibodies are b oth derived from the same variable region germline genes but differ in affinity due to somatic mutations present in 36-71. To investigate th e poor expression of 36-71 Fab, we examined several factors, such as c ellular toxicity, induction with isopropylthio-beta-D-galactoside, and growth of transformed bacteria at lower temperatures (30 degrees C), as well as the possibility of E. coli strain-related expression of Fab . However, none of these factors made a significant difference to Fab expression. We next localized a significant portion of the defect in F ab expression to the heavy chain by swapping the heavy and light chain s from the two antibodies to construct hybrid Fabs. We used site-direc ted mutagenesis to engineer amino acids into the variable regions of a ntibody 36-71, to reproduce those found in 36-65 which is expressed we ll in E. coli. The defect in expression is due to residues located in the complementarity-determining regions, as mutations of heavy chain f ramework residues to those present in 36-65 do not enhance expression of 36-71 Fab in E. coli.