Haimovitz Rachel, Barak Yoav, Morag Ely, Voronov-Goldman Milana, Shoham Yuval, Lamed Raphael, Bayer Edward A
Department of Biological Chemistry, The Weizmann Institute of Science, Rehovot, Israel.
Proteomics. 2008 Mar;8(5):968-79. doi: 10.1002/pmic.200700486.
The cellulosome is an intricate multienzyme complex, designed for efficient degradation of plant cell wall polysaccharides, notably cellulose. The supramolecular cellulosome architecture in different bacteria is the consequence of the types and specificities of the interacting cohesin and dockerin modules, borne by the different cellulosomal subunits. In this study, we describe a microarray system for determining cohesin-dockerin specificity, which allows global comparison among the interactions between various members of these two complementary families of interacting protein modules. Matching recombinant fusion proteins were prepared that contained one of the interacting modules: cohesins were joined to an appropriate cellulose-binding module (CBM) and the dockerins were fused to a thermostable xylanase that served to enhance expression and proper folding. The CBM-fused cohesins were immobilized on cellulose-coated glass slides, to which xylanase-fused dockerin samples were applied. Knowledge of the specificity characteristics of native and mutated members of the cohesin and dockerin families provides insight into the architecture of the parent cellulosome and allows selection of suitable cohesin-dockein pairs for biotechnological and nanotechnological application. Using this approach, extensive cross-species interaction among type-II cohesins and dockerins is shown for the first time. Selective intraspecies binding of an archaeal dockerin to two complementary cohesins is also demonstrated.
纤维小体是一种复杂的多酶复合体，旨在高效降解植物细胞壁多糖，尤其是纤维素。不同细菌中的超分子纤维小体结构是由不同纤维小体亚基所携带的相互作用的黏附素和锚定蛋白模块的类型和特异性决定的。在本研究中，我们描述了一种用于确定黏附素 - 锚定蛋白特异性的微阵列系统，该系统允许对这两个相互作用的互补蛋白模块家族的各个成员之间的相互作用进行全面比较。制备了匹配的重组融合蛋白，其中包含一个相互作用模块：将黏附素与合适的纤维素结合模块（CBM）连接，将锚定蛋白与热稳定木聚糖酶融合，以增强表达和正确折叠。将与CBM融合的黏附素固定在涂有纤维素的载玻片上，然后将与木聚糖酶融合的锚定蛋白样品应用于其上。了解黏附素和锚定蛋白家族天然成员和突变成员的特异性特征，有助于深入了解亲本纤维小体的结构，并允许选择适合生物技术和纳米技术应用的黏附素 - 锚定蛋白对。使用这种方法，首次展示了II型黏附素和锚定蛋白之间广泛的跨物种相互作用。还证明了古菌锚定蛋白与两种互补黏附素的选择性种内结合。
