BACTERIAL ASPECTS ASSOCIATED WITH THE EXPRESSION OF A SINGLE-CHAIN ANTIBODY FRAGMENT IN ESCHERICHIA-COLI

The bacterial expression of a single-chain antibody fragment, designat ed L6 sFv, was examined. Periplasmic targeting resulted in the product ion of a correctly folded protein that bound tumor antigen. However, i mmediately after induction at either 30 degrees C or 37 degrees C ther e was a significant loss in bacterial viability, which was followed by a loss in absorbance. The loss in absorbance correlated with cell lys is and release of the L6 sFv into the culture supernatant. The kinetic s of appearance of L6 sFv in the supernatant paralleled that of peripl asmic beta-lactamase and confirmed an initial loss of cell-wall integr ity prior to cell lysis. Bacteria incubated at 30 degrees C produced a pproximately threefold more correctly folded antibody fragment because of an increase in the number of cells/A(660) at the lower incubation temperature. More than 95% of the L6 sFv, made at either incubation te mperature, was incorrectly folded. Osmotic-shock procedures did not re lease L6 sFv. However, in situ subtilisin susceptibility experiments w ith bacterial spheroplasts confirmed a periplasmic location. French pr ess disruption resulted in the release of correctly but not incorrectl y folded material. Membrane fractionation revealed that the incorrectl y folded L6 sFv remained associated with both the inner and outer memb rane. These results demonstrate that, in this system, antibody fragmen t expression resulted initially in cell death, which was followed by r elease of protein into the culture supernatant and eventually cell lys is. It is also suggested that membrane association in the periplasmic space may impede proper folding.