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.
Department of Chemical Engineering, University of Waterloo, ON, Canada.
R S
RS
Donovan
C W
CW
Robinson
B R
BR
Glick
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