RESEARCH ARTICLE Cohesin-dockerin microarray: Diverse specificities between two complementary families of interacting protein modules Rachel Haimovitz 1 * , Yoav Barak 1 * , Ely Morag 2 , Milana Voronov-Goldman 3 , Yuval Shoham 4 , Raphael Lamed 3 and Edward A. Bayer 1 1 Department of Biological Chemistry, The Weizmann Institute of Science, Rehovot, Israel 2 Zephyr ProteomiX, Kiryat-Shmona, Israel 3 Department of Molecular Microbiology and Biotechnology, Tel Aviv University, Ramat Aviv, Israel 4 Department of Biotechnology and Food Engineering, and Institute of Catalysis Science and Technology, Technion–Israel Institute of Technology, Haifa,Israel The cellulosome is an intricate multienzyme complex, designed for efficient degradation of plant cell wall polysaccharides, notably cellulose. The supramolecular cellulosome architecture in dif- ferent 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 con- tained 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. Received: May 31, 2007 Revised: November 19, 2007 Accepted: November 21, 2007 Keywords: Cellulose-binding module (CBM) / Cellulose-coated glass slide / Cohesin-dockerin recognition / Protein microarray 968 Proteomics 2008, 8, 968–979 1 Introduction Cellulosomes are multicomponent, multienzyme machines, produced by anaerobic bacteria for the efficient digestion of cellulose and other plant cell wall polysaccharides [1–3]. The various enzymes and other cellulosome components are Correspondence: Professor Edward A. Bayer, Department of Bio- logical Chemistry, The Weizmann Institute of Science, Rehovot 76100, Israel E-mail: ed.bayer@weizmann.ac.il Fax: 1972-8-946-8256 Abbreviations: CBM, cellulose-binding module; CBM-Coh, CBM- fused cohesin; SPR, surface plasmon resonance; Xyn, xylanase; XynDoc, xylanase-fused dockerin * Both these authors have contributed equally to this work. DOI 10.1002/pmic.200700486  2008 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.proteomics-journal.com