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waa P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Waa P蛋白,并利用亲合层析和凝胶过滤层析对可溶性Waa P蛋白进行纯化,用SDS-PAGE进行检测。对比大肠杆菌和铜绿假单胞菌中Waa P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Waa P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Waa P,为蛋白结构解析时相位的确定提供依据。
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 Waa Ps from these two strains were compared and analyzed. We optimized procedure to obtain high-yield and pure Waa P protein for crystal screening. Selenomethionine WaaP was also expressed to provide phasing for structure determination.
出处
《化学通报》
CAS
CSCD
北大核心
2014年第12期1196-1201,共6页
Chemistry
关键词
抗生素
革兰氏阴性菌
脂多糖
外膜
WaaP
Antibiotic
Gram-negative bacteria
Lipopolysaccharide
Outer membrane
WaaP
