INTRACELLULAR AND EXTRACELLULAR EXPRESSION OF AN SCFV ANTIBODY FRAGMENT IN ESCHERICHIA-COLI - EFFECT OF BACTERIAL STRAINS AND PATHWAY ENGINEERING USING GROES L CHAPERONINS/

We have studied the influence of bacterial host on the secretion of si ngle-chain Fv antibody fragment (scFv), the production of this antibod y fragment as intracellular fusion protein, and the effect of chaperon in coexpression on intracellular antibody expression. Seven bacterial strains were transformed with a vector carrying the genes encoding the variable regions of an anti-CEA scFv antibody and the ompA leader seq uence (ptrp/ompA/scFvCEA). Expression and secretion of this antibody f ragment were highest in the W3110 strain, as determined by Western blo t analysis and enzyme immnnoassay, where the scFv fragment amounted to approximately 30% of the total periplasmic protein. Except for BMH71- 18, the other strains were unsuitable for antibody fragment expression , suggesting screening of bacterial strains as an important parameter For intracellular expression, the scFv was expressed as a fusion prote in with a 26-amino acid N-terminal fragment of human interleukin-2 (IL -2), using the pIL-2f/scFvCEA vector: The fusion protein was expressed at 30% of total biomass and retained antigen binding after in vitro r efolding. Co-expression of chaperonin-encoding plasmid pGroES/L with p IL-2f/scFv increased the intracellular- production of the fusion prote in two-fold with a similar increase in the final amount of active scFv antibody fragment that could be obtained after in vitro refolding. Th e chaperonins had no effect on secretion of scFv antibody fragments, u sing the ptrp/ompA/scFvCEA.