Madduri Krishna, Badger Monty, Li Ze-Sheng, Xu Xiaoping, Thornburgh Scott, Evans Steve, Dhadialla Tarlochan S
Dow AgroSciences LLC, 9330 Zionsville Road, Indianapolis, IN 46268, USA.
Protein Expr Purif. 2009 May;65(1):57-65. doi: 10.1016/j.pep.2008.12.012. Epub 2008 Dec 30.
Pseudomonas fluorescens is a robust protein expression system that is very well suited for high throughput protein expression for structural genomics studies. Since NMR spectroscopy and X-ray crystallography are both used by various investigators in structure elucidation studies, the availability of target proteins labeled with stable isotopes or selenomethionine is essential for the determination of protein structures. A completely defined medium for the expression and stable isotope labeling of proteins in P. fluorescens has been developed. The expression level of Bacillus thuringiensis Cry34 in the modified medium is comparable to that obtained in the original medium. In addition, more than 95% incorporation of 15N was obtained in Cry34 using 15N ammonium sulfate and the quality of the protein, as assessed by NMR analysis, is comparable to that made using commercial medium. High levels of selenomethionine (SeMet) incorporation in the Xenorhabdus nematophilus insecticidal protein XptA2 were also obtained in P. fluorescens using the defined medium, allowing development of a method for obtaining highly purified XptA2. The following observations were made when inhibitors of endogenous methionine biosynthesis were used in P. fluorescens culture when SeMet was substituted in XptA2: (I) there is little inhibition of cell growth or recombinant XptA2 expression in the presence of SeMet concentrations up to 300 mg/L in cell culture, (II) there was greater than 95% SeMet incorporation ratio in recombinant SeMet-labeled XptA2 (SeMet-XptA2) and the incorporation ratio is consistent and reproducible and (III) finally, purified SeMet-XptA2 possesses similar protein structure and insecticidal activity relative to the unlabeled counterpart XptA2 as shown by bioassay and differential scanning calorimetric analysis. The high SeMet incorporation should provide high accuracy and resolution in XptA2 phase determination by multiwavelength anomalous diffraction (MAD), indicating that P. fluorescens is an excellent expression host to produce SeMet-labeled proteins for structural study.
荧光假单胞菌是一种强大的蛋白质表达系统，非常适合用于结构基因组学研究的高通量蛋白质表达。由于核磁共振光谱法和X射线晶体学在结构解析研究中都被不同的研究人员所使用，因此获得用稳定同位素或硒代甲硫氨酸标记的目标蛋白质对于蛋白质结构的测定至关重要。已经开发出一种用于荧光假单胞菌中蛋白质表达和稳定同位素标记的完全确定的培养基。苏云金芽孢杆菌Cry34在改良培养基中的表达水平与在原始培养基中获得的表达水平相当。此外，使用15N硫酸铵在Cry34中获得了超过95%的15N掺入率，通过核磁共振分析评估，该蛋白质的质量与使用商业培养基制备的蛋白质质量相当。使用确定的培养基在荧光假单胞菌中也获得了嗜线虫致病杆菌杀虫蛋白XptA2中高水平的硒代甲硫氨酸（SeMet）掺入，从而开发出一种获得高度纯化的XptA2的方法。当在XptA2中用SeMet替代时，在荧光假单胞菌培养中使用内源性甲硫氨酸生物合成抑制剂时，有以下观察结果：（I）在细胞培养中SeMet浓度高达300 mg/L的情况下，细胞生长或重组XptA2表达几乎没有受到抑制，（II）重组SeMet标记的XptA2（SeMet-XptA2）中的SeMet掺入率大于95%，并且掺入率是一致且可重复的，（III）最后，如生物测定和差示扫描量热分析所示，纯化的SeMet-XptA2相对于未标记的对应物XptA2具有相似的蛋白质结构和杀虫活性。高SeMet掺入率应能在通过多波长反常衍射（MAD）进行的XptA2相位测定中提供高精度和分辨率，表明荧光假单胞菌是用于结构研究生产SeMet标记蛋白质的优秀表达宿主。
