Architecture et Fonction des Macromolécules Biologiques (AFMB), UMR 6098 CNRS and Universités Aix-Marseille I & II, Campus de Luminy, Case 932, Marseille Cedex 09, France.
J Struct Biol. 2010 Oct;172(1):75-84. doi: 10.1016/j.jsb.2010.02.007. Epub 2010 Feb 11.
We report here the characterization of several large structural protein complexes forming the baseplates (or part of them) of Siphoviridae phages infecting Lactococcus lactis: TP901-1, Tuc2009 and p2. We revisited a "block cloning" expression strategy and extended this approach to genomic fragments encoding proteins whose interacting partners have not yet been clearly identified. Biophysical characterization of some of these complexes using circular dichroism and size exclusion chromatography, coupled with on-line light scattering and refractometry, demonstrated that the over-produced recombinant proteins interact with each other to form large (up to 1.9MDa) and stable baseplate assemblies. Some of these complexes were characterized by electron microscopy confirming their structural homogeneity as well as providing a picture of their overall molecular shapes and symmetry. Finally, using these results, we were able to highlight similarities and differences with the well characterized much larger baseplate of the myophage T4.
我们在此报告了几种形成感染乳球菌的肌尾噬菌体基板（或基板的一部分）的大型结构蛋白复合物的特征：TP901-1、 Tuc2009 和 p2。我们重新审视了一种“块克隆”表达策略，并将该方法扩展到编码其相互作用伙伴尚未明确鉴定的蛋白质的基因组片段。使用圆二色性和尺寸排阻色谱法对其中一些复合物进行的生物物理特性分析，结合在线光散射和折射计，证明过量表达的重组蛋白相互作用形成大的（高达 1.9MDa）且稳定的基板组装体。通过电子显微镜对其中一些复合物进行了表征，证实了它们的结构均一性，并提供了它们整体分子形状和对称性的图像。最后，利用这些结果，我们能够突出与特征良好的肌尾噬菌体 T4 更大基板的相似之处和差异。
