Optimizing the expression of a monoclonal antibody fragment under the transcriptional control of the Escherichia coli lac promoter.
The expression of a monoclonal antibody Fab fragment in Escherichia coli strain RB791/pComb3, induced with either lactose or isopropyl-beta-D-thiogalactoside (IPTG), was compared to determine if lactose might provide an inexpensive alternative to induction with IPTG. Induction of Fab expression imposed a metabolic load on the recombinant cells, resulting in lower final cell yields compared to the non-induced controls. An IPTG concentration of 0.05 mM was sufficient to achieve maximal expression of soluble Fab protein when inducing in the early-, mid-, or late-log phases of batch cultures grown using either glucose or glycerol as a carbon source. The largest overall yield of Fab fragments when using 0.05 mM IPTG was achieved by increasing the final yield of cells through glycerol feeding following induction in late-log phase. Lactose was as effective as IPTG for inducing Fab expression in E. coli RB791/pComb3. The greatest overall level of Fab expression was found when cells grown on glycerol were induced with 2 g/L lactose in late-log phase. Since the cost of 0.05 mM of IPTG is significantly greater than the cost of 2 g/L lactose, lactose provides an inexpensive alternative to IPTG for inducing the expression of Fab fragments, and possibly other recombinant proteins, from the E. coli lac promoter.
Donovan
R S
RS
Department of Chemical Engineering, University of Waterloo, ON, Canada.
Robinson
C W
CW
Glick
B R
BR
D000911:Antibodies, Monoclonal / Q000096:biosynthesis*; D015153:Blotting, Western; D003470:Culture Media; D004926:Escherichia coli / Q000235:genetics* / Q000254:growth & development / Q000378:metabolism; D005947:Glucose / Q000378:metabolism; D005990:Glycerol / Q000378:metabolism; D007140:Immunoglobulin Fab Fragments / Q000096:biosynthesis*; D007544:Isopropyl Thiogalactoside / Q000494:pharmacology; D007763:Lac Operon; D007785:Lactose / Q000494:pharmacology; D011401:Promoter Regions, Genetic; D011994:Recombinant Proteins / Q000096:biosynthesis; D015533:Transcriptional Activation; D001616:beta-Galactosidase / Q000378:metabolism
D000911:Antibodies, Monoclonal; D003470:Culture Media; D007140:Immunoglobulin Fab Fragments; D011994:Recombinant Proteins; D007544:Isopropyl Thiogalactoside; D001616:beta-Galactosidase; D005947:Glucose; D007785:Lactose; D005990:Glycerol