Factors affecting the fermentative production of a lysozyme-binding antibody fragment in Escherichia coli.
Fed-batch fermentation for production of a single-chain Fv antibody fragment (scFv) expressed as a recombinant periplastic protein from Escherichia coli was investigated. A high cell density of 50 g dry cell weight per liter was routinely achieved in a 14-L vessel by controlled exponential feeding of glucose to impose a constant specific growth rate. Following biomass accumulation, induction of the tac promoter by addition of IPTG was accompaied by a linear feed of yeast extract. The concentration of yeast extract feed was found to be highly influential upon both concentration and location of active product. Although scFv fragments were specifically targeted to the periplasmic space, at yeast extract feed rates of 0.72 g/h the final location was largely extracellular (68% to 79%). Total concentrations (extracellular + periplasmic) were of the order of 5 to 8 mg/L. A ten-fold increase in yeast extract supply increased total scFv concentration to almost 200 mg/L and 78% of this yield was retained in the periplasm. Control of such leakage of the recombinant product is fundamental to process design of downstream operations for product recovery.
Harrison
J S
JS
Department of Chemical and Biochemical Engineering, The Advanced Centre for Biochemical Engineering, University College London, Torrington Place, London WC1E 7JE, UK. j. harrison@ucl.ac.uk
Keshavarz-Moore
E
E
Dunnill
P
P
Berry
M J
MJ
Fellinger
A
A
Frenken
L
L