Heterogenous Expression and Purification of Lipid II Flippase from Staphylococcus aureus

Area of Science:

• Microbiology
• Structural Biology
• Drug Discovery

Background:

• *Staphylococcus aureus* poses a significant threat due to antibiotic resistance.
• MurJ (SaMurJ), a Lipid II flippase in *S. aureus*, is a promising target for novel antibacterial agents.
• Efficient expression and purification of SaMurJ are crucial for drug development.

Purpose of the Study:

• Optimize SaMurJ expression and purification.
• Identify optimal detergents for SaMurJ extraction and solubilization.
• Investigate the peptidisc system for creating a detergent-free SaMurJ environment.

Main Methods:

• SaMurJ with an N-terminal ten-His tag was expressed without induction.
• Six detergents were screened for protein extraction and solubilization efficiency.
• Thermostability was assessed, and peptidisc assembly was attempted.

Main Results:

• SaMurJ was successfully expressed in BL21(DE3) with a yield of 1 mg/L.
• DDM was identified as the optimal detergent for SaMurJ solubilization.
• Purification via nickel affinity chromatography yielded ~88% pure SaMurJ, but NSPr did not stabilize the protein.

Conclusions:

• High-purity SaMurJ was successfully expressed and purified.
• DDM-containing buffer effectively solubilizes and stabilizes SaMurJ.
• Further research is needed to stabilize SaMurJ in a detergent-free system.