生产,提取,净化,和稳定完整的膜蛋白是成功的结构生物学研究的关键步骤，特别是X射线晶体学或单粒子显微镜。这里,我们提出了从铜绿假单胞菌中纯化CntI的方案，药物代谢转运蛋白（DMT）家族的一个新的金属矿出口国参与假嘌呤分泌。在CntI纯化之后，我们优化了缓冲液的pH值,盐，和添加剂的差示扫描荧光分析（DSF），也称为热荧光测定（TFA）或荧光热稳定性测定（FTSA），使用染料1-苯胺萘-8-磺酸（ANS），与洗涤剂相容的荧光分子。缓冲区优化后，纯化的CntI通过尺寸排阻色谱法与多角度激光散射（SEC-MALLS）进行分析，UV吸光度,和折射率检测器，为了确定蛋白质-洗涤剂复合物的绝对摩尔质量，与蛋白质结合的洗涤剂量和无蛋白质洗涤剂胶束的量。总之,这些生物物理技术提供了有关纯化膜蛋白是否适合进一步生物物理或结构研究的初步和强制性信息。
Production, extraction, purification, and stabilization of integral membrane proteins are key steps for successful structural biology studies, in particular for X-ray crystallography or single particle microscopy. Here, we present the purification protocol of CntI from Pseudomonas aeruginosa, a new metallophore exporter of the Drug Metabolite Transporter (DMT) family involved in pseudopaline secretion. Subsequent to CntI purification, we optimized the buffer pH, salts, and additives by differential scanning fluorimetry (DSF), also known as Thermofluor Assay (TFA) or fluorescent thermal stability assay (FTSA), with the use of dye 1-AnilinoNaphthalene-8-Sulfonic acid (ANS), a fluorescent molecule compatible with detergents. After the buffer optimization, the purified CntI was analyzed by Size Exclusion Chromatography coupled with Multi-Angle Laser Light Scattering (SEC-MALLS), UV absorbance, and Refractive Index detectors, in order to determine the absolute molar mass of the protein-
detergent complex, the
detergent amount bound to the protein and the amount of protein-free
detergent micelles. Altogether, these biophysical techniques give preliminary and mandatory information about the suitability of the purified membrane protein for further biophysical or structural investigations.
