%0 Journal Article
%T Heterogenous Expression and Purification of Lipid II Flippase from Staphylococcus aureus.
%A Zheng YY
%A Chung WH
%A Leung YC
%A Wong KY
%J Protein Pept Lett
%V 31
%N 5
%D 2024
%M 38967080
%F 1.927
%R 10.2174/0109298665316374240531113258
%X <B>BACKGROUND: </B>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.<BR><B>OBJECTIVE: </B>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.<BR><B>METHODS: </B>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.<BR><B>RESULTS: </B>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.<BR><B>CONCLUSIONS: </B>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.