| 金瑾,朱嘉,杨少媛,雷振,郑积敏,贾宗超.化学通报,2014,77(12):1196-1201. |
| 革兰氏阴性菌WaaP蛋白的表达与纯化对比研究 |
| Comparation Studies of Expression and Purification of WaaP proteins from Gram-negative Bacteria |
| 投稿时间：2014-05-22 修订日期：2014-06-20 |
| DOI： |
| 中文关键词: 抗生素 革兰氏阴性菌 脂多糖 外膜 WaaP |
| 英文关键词:Antibiotic Gram-negative bacteria Lipopolysaccharide Outer membrane WaaP |
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| 中文摘要: |
| 采用基因重组方法构建来源于大肠杆菌和铜绿假单胞菌的waaP基因的克隆，利用多种感受态细胞表达出带有不同纯化标签的可溶性WaaP蛋白，并利用亲合层析和凝胶过滤层析对可溶性WaaP蛋白进行纯化，并用 SDS-PAGE进行检测。对比大肠杆菌和铜绿假单胞菌中WaaP的表达和纯化结果，为蛋白结晶选取能够得到大量稳定和高纯度WaaP蛋白的表达纯化方法，并用该方法，使用硒代甲硫氨酸培养基表达出硒代甲硫氨酸标记的WaaP，为蛋白结构解析时相位的确定提供依据。 |
| 英文摘要: |
| In this study, recombinant DNA technology was used to produce clones harboring waaP genes from Escherichia coli and Pseudomonas aeruginosa. WaaP proteins from these two strains were expressed in various competent cells and fused with different tags for purification. The soluble WaaP proteins were purified by affinity chromatography and gel filtration chromatography, then detected with SDS-PAGE. The results of WaaPs from these two strains were compared and analyzed. We optimized procedure to obtain high-yield and pure WaaP protein for crystal screening. Selenomethionine WaaP was also expressed to provide phasing for structure determination. |
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