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Updated: Aug 17, 2026
A Fluorescence-based Assay of Phospholipid Scramblase Activity
Published on: September 20, 2016
Heterogenous Expression and Purification of Lipid II Flippase from Staphylococcus aureus
Yuan Yuan Zheng1, Wai-Hong Chung1, Yun-Chung Leung1
1State Key Laboratory of Chemical Biology and Drug Discovery, Department of Applied Biology and Chemical Technology, The Hong Kong Polytechnic University, Hung Hom, Hong Kong, China.
Background:
Staphylococcus aureus is a common pathogen with strains that are resistant to existing antibiotics. MurJ from S. aureus (SaMurJ), an integral membrane protein functioning as Lipid II flippase, is a potential target for developing new antibacterial agents against this pathogen. Successful expression and purification of this protein shall be useful in the development of drugs against this target.
Objective:
In this study, we demonstrated the optimized expression and purification procedures of SaMurJ, identified suitable detergent for extracting and solubilizing the protein, and examined the peptidisc system to generate a detergent-free environment.
Methods:
SaMurJ fused with N-terminal ten-His tag was expressed without induction. Six detergents were selected for screening the most efficient candidate for extraction and solubilization of the protein. The thermostability of the detergent-solubilized protein was assessed by evaluated temperature incubation. Different ratios of peptidisc bi-helical peptide (NSPr) to SaMurJ were mixed and the on-bead peptidisc assembly method was applied.
Results:
SaMurJ expressed in BL21(DE3) was confirmed by peptide fingerprinting, with a yield of 1 mg SaMurJ per liter culture. DDM was identified as the optimum detergent for solubilization and the nickel affinity column enabled SaMurJ purification with a purity of ~88%. However, NSPr could not stabilize SaMurJ.
Conclusion:
The expression and purification of SaMurJ were successful, with high purity and good yield. SaMurJ can be solubilized and stabilized by a DDM-containing buffer.
Insights
We successfully expressed and purified Staphylococcus aureus MurJ (SaMurJ), a potential antibiotic target. SaMurJ can be solubilized using DDM detergent, aiding in the development of new antibacterial drugs.
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.
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