The E. coli pET expression system revisited-mechanistic correlation between glucose and lactose uptake

Area of Science:

• Biotechnology
• Molecular Biology
• Bioprocess Engineering

Background:

• Therapeutic monoclonal antibodies are typically produced in mammalian cells.
• Escherichia coli offers advantages for producing unglycosylated antibody fragments, including cost-effective media and high productivity.
• The pET expression system in E. coli BL21(DE3) is common but isopropyl β-D-1-thiogalactopyranoside (IPTG) induction can cause cell stress and insoluble protein formation.

Purpose of the Study:

• To optimize the production of soluble antibody single-chain variable fragments (scFv) using the pET expression system in E. coli.
• To compare lactose induction with IPTG induction for soluble scFv production.
• To investigate the influence of specific glucose uptake rate (q s,glu) on soluble product formation during lactose induction and to model the correlation between lactose (q s,lac) and glucose (q s,glu) uptake rates.

Main Methods:

• Recombinant production of a novel antibody scFv in E. coli BL21(DE3) using the pET expression system.
• Comparative induction studies using lactose versus isopropyl β-D-1-thiogalactopyranoside (IPTG).
• Controlled fermentation experiments to vary specific glucose uptake rate (q s,glu) during lactose induction.
• Development of a mechanistic model correlating specific lactose uptake rate (q s,lac) with q s,glu.

Main Results:

• Lactose induction yielded significantly higher amounts of soluble scFv compared to IPTG induction, even at increased growth rates.
• The specific glucose uptake rate (q s,glu) during lactose induction was found to effectively tune the production of soluble scFv.
• A mechanistic model was established to describe the relationship between specific lactose uptake rate (q s,lac) and specific glucose uptake rate (q s,glu), enabling controlled feeding strategies.

Conclusions:

• Lactose is a superior inducer for soluble scFv production in E. coli using the pET system compared to IPTG.
• Controlling the specific glucose uptake rate during lactose induction allows for optimization of soluble recombinant protein yield.
• The developed mechanistic model provides a foundation for tailored feeding strategies and prevention of sugar accumulation in E. coli-based bioprocesses.