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.
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.
Horn
U
U
Hans-Knöll-Institut für Naturstoff-Forschung, Jena, Germany.
Strittmatter
W
W
Krebber
A
A
Knüpfer
U
U
Kujau
M
M
Wenderoth
R
R
Müller
K
K
Matzku
S
S
Plückthun
A
A
Riesenberg
D
D
D000085:Acetates / Q000378:metabolism; D000917:Antibody Formation; D003001:Cloning, Molecular; D018388:Codon, Terminator; D003470:Culture Media / Q000737:chemistry / Q000378:metabolism; D066246:ErbB Receptors / Q000235:genetics* / Q000276:immunology*; D004926:Escherichia coli / Q000235:genetics* / Q000254:growth & development; D005285:Fermentation / Q000235:genetics; D015964:Gene Expression Regulation, Bacterial; D005822:Genetic Vectors / Q000235:genetics* / Q000502:physiology*; D008967:Molecular Biology / Q000379:methods*
D000085:Acetates; D018388:Codon, Terminator; D003470:Culture Media; D066246:ErbB Receptors