Bacterial aspects associated with the expression of a single-chain antibody fragment in Escherichia coli.
The bacterial expression of a single-chain antibody fragment, designated L6 sFv, was examined. Periplasmic targeting resulted in the production of a correctly folded protein that bound tumor antigen. However, immediately after induction at either 30 degrees C or 37 degrees C there was a significant loss in bacterial viability, which was followed by a loss in absorbance. The loss in absorbance correlated with cell lysis and release of the L6 sFv into the culture supernatant. The kinetics of appearance of L6 sFv in the supernatant paralleled that of periplasmic beta-lactamase and confirmed an initial loss of cell-wall integrity prior to cell lysis. Bacteria incubated at 30 degrees C produced approximately threefold more correctly folded antibody fragment because of an increase in the number of cells/A660 at the lower incubation temperature. More than 95% of the L6 sFv, made at either incubation temperature, was incorrectly folded. Osmotic-shock procedures did not release L6 sFv. However, in situ subtilisin susceptibility experiments with bacterial spheroplasts confirmed a periplasmic location. French press disruption resulted in the release of correctly but not incorrectly folded material. Membrane fractionation revealed that the incorrectly folded L6 sFv remained associated with both the inner and outer membrane. These results demonstrate that, in this system, antibody fragment expression resulted initially in cell death, which was followed by release of protein into the culture supernatant and eventually cell lysis. It is also suggested that membrane association in the periplasmic space may impede proper folding.
Somerville
J E
JE
Inflammation Department, Bristol-Myers Squibb Pharmaceutical Research Institute, Seattle, WA 98121.
Goshorn
S C
SC
Fell
H P
HP
Darveau
R P
RP
D000595:Amino Acid Sequence; D000818:Animals; D001709:Biotechnology; D004926:Escherichia coli / Q000235:genetics* / Q000276:immunology*; D015870:Gene Expression; D007128:Immunoglobulin Fragments / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics*; D008969:Molecular Sequence Data; D015202:Protein Engineering; D017510:Protein Folding; D013347:Subcellular Fractions / Q000276:immunology
D007128:Immunoglobulin Fragments