Article,
High volumetric yields of functional dimeric miniantibodies in Escherichia coli, using an optimized expression vector and high-cell-density fermentation under non-limited growth conditions.
Appl Microbiol Biotechnol, 46 (5-6): 524--532 (December 1996)
Abstract
Functional bivalent miniantibodies, directed against the epidermal
growth factor receptor, accumulated to more than 3 gl-1 in high-cell-density
cultures of Escherichia coli RV308(pHKK) on a pilot scale. The miniantibodies
consist of scFv fragments with a C-terminal hinge followed by a
helix-turn-helix motif, which homodimerizes in vivo. The improved
expression vector pHKK is characterized by the hoklsok suicide system,
improving plasmid maintenance, and the inducible lac pl o promoter
system with the very strong T7g10 Shine-Dalgarno sequence. The expression
unit is flanked by terminators. The prototrophic RV308 cells were
cultivated in glucose mineral salt medium and reached a cell density
of 145 g dry biomass l-1 after 33 h. After induction, growth continued
almost unchanged for a further 4 h with concomitant miniantibody
formation. In the fedbatch phase, the concentration of glucose was
kept almost constant at the physiological level of approximately
1.5 gl-1, using on-line flow injection analysis for control. Surprisingly,
E. coli RV308(pHKK) did not accumulate significant amounts of the
metabolic by-product acetate under these unlimited aerobic growth
conditions.
