Abstract：
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.
CONCLUSIONS: 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.
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.
CONCLUSIONS: 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.
摘要：
背景：金黄色葡萄球菌是一种常见的病原体，其菌株对现有抗生素具有抗性。来自金黄色葡萄球菌的MurJ（SaMurJ），一种作为脂质II翻转酶的完整膜蛋白，是开发针对这种病原体的新型抗菌剂的潜在目标。该蛋白质的成功表达和纯化将有助于开发针对该靶标的药物。
目的：在本研究中，我们展示了SaMurJ的优化表达和纯化程序，确定了用于提取和溶解蛋白质的合适洗涤剂，并检查了peptidisc系统以产生无洗涤剂的环境。
方法:与N-末端10-His标签融合的SaMurJ无诱导表达。选择六种去污剂来筛选用于蛋白质提取和溶解的最有效候选物。通过评估的温度孵育来评估洗涤剂溶解的蛋白质的热稳定性。将不同比例的肽盘双螺旋肽(NSPr)与SaMurJ混合,并应用珠上肽盘组装方法。
结果：SaMurJ在BL21（DE3）中的表达通过肽指纹图谱得到证实，每升培养物的产量为1毫克SaMurJ。DDM被确定为用于溶解的最佳去污剂,并且镍亲和柱使得SaMurJ纯化具有〜88%的纯度。然而,NSPr不能稳定SaMurJ。
结论：SaMurJ的表达和纯化是成功的，纯度高，收率好。SaMurJ可以通过含DDM的缓冲液溶解和稳定。
目的：在本研究中，我们展示了SaMurJ的优化表达和纯化程序，确定了用于提取和溶解蛋白质的合适洗涤剂，并检查了peptidisc系统以产生无洗涤剂的环境。
方法:与N-末端10-His标签融合的SaMurJ无诱导表达。选择六种去污剂来筛选用于蛋白质提取和溶解的最有效候选物。通过评估的温度孵育来评估洗涤剂溶解的蛋白质的热稳定性。将不同比例的肽盘双螺旋肽(NSPr)与SaMurJ混合,并应用珠上肽盘组装方法。
结果：SaMurJ在BL21（DE3）中的表达通过肽指纹图谱得到证实，每升培养物的产量为1毫克SaMurJ。DDM被确定为用于溶解的最佳去污剂,并且镍亲和柱使得SaMurJ纯化具有〜88%的纯度。然而,NSPr不能稳定SaMurJ。
结论：SaMurJ的表达和纯化是成功的，纯度高，收率好。SaMurJ可以通过含DDM的缓冲液溶解和稳定。
