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Matching fusion protein systems for affinity analysis of two interacting families of proteins: the cohesin-dockerin interaction
Authors:Barak Yoav  Handelsman Tal  Nakar David  Mechaly Adva  Lamed Raphael  Shoham Yuval  Bayer Edward A
Affiliation:Department of Biological Chemistry, The Weizmann Institute of Science, Rehovot 76100, Israel.
Abstract:Cellulosomes are multi-enzyme complexes that orchestrate the efficient degradation of cellulose and related plant cell wall polysaccharides. The complex is maintained by the high-affinity protein-protein interaction between two complementary modules: the cohesin and the dockerin. In order to characterize the interaction between different cohesins and dockerins, we have developed matching fusion-protein systems, which harbor either the cohesin or the dockerin component. For this purpose, corresponding plasmid cassettes were designed, which encoded for the following carrier proteins: (i) a thermostable xylanase with an appended His-tag; and (ii) a highly stable cellulose-binding module (CBM). The resultant xylanase-dockerin and CBM-cohesin fusion products exhibited high expression levels of soluble protein. The expressed, affinity-purified proteins were extremely stable, and the functionality of the cohesin or dockerin component was retained. The fusion protein system was used to establish a sensitive and reliable, semi-quantitative enzyme-linked affinity assay for determining multiple samples of cohesin-dockerin interactions in microtiter plates. A variety of cohesin-dockerin systems, which had been examined previously using other methodologies, were revisited applying the affinity-based enzyme assay, the results of which served to verify the validity of the approach.
Keywords:protein–protein interactions  modular proteins  multi‐protein complex  cellulosome  ELISA  protein families
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