Human DT-diaphorase expression in Escherichia coli: optimization, purification and structural stability

Area of Science:

• Biochemistry
• Enzyme kinetics
• Protein engineering

Background:

• Human DT-diaphorase (NAD(P)H quinone oxidoreductase 1, NQO1) is a flavoprotein crucial for biosensor applications, including glucose level monitoring.
• Optimizing its expression and understanding its stability are key for reliable biosensor design.

Purpose of the Study:

• To optimize the expression and purification of human DT-diaphorase in Escherichia coli.
• To investigate the thermal stability of DT-diaphorase activity in the presence of varying sucrose concentrations.

Main Methods:

• Optimized expression conditions in E. coli BL21 (DE3) using specific induction time, temperature, lactose, and IPTG concentrations.
• Assessed enzyme kinetics (Km, Vmax, kcat) for NADH.
• Evaluated DT-diaphorase activity and structural changes at different temperatures (40°C and 50°C) with varying sucrose concentrations (0.75 M).

Main Results:

• Optimal expression achieved with specific induction parameters: 22 hr induction time, 18°C induction temperature, 5 mM lactose, and 1 mM IPTG.
• Kinetic parameters for NADH were determined: Km = 25.50 µM, Vmax = 357 µM/min, kcat = 446.40 μM mg⁻¹ min⁻¹.
• Sucrose (0.75 M) at 50°C significantly reduced DT-diaphorase inactivation rate, offering moderate thermal stabilization and preserving enzyme conformation.

Conclusions:

• Established optimized conditions for high-level expression and purification of active human DT-diaphorase.
• Demonstrated that sucrose enhances DT-diaphorase thermal stability, making it suitable for applications requiring elevated temperatures.