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Matching fusion protein systems for affinity analysis of two interacting families of proteins: the cohesin–dockerin interaction
Author(s) -
Barak Yoav,
Handelsman Tal,
Nakar David,
Mechaly Adva,
Lamed Raphael,
Shoham Yuval,
Bayer Edward A.
Publication year - 2005
Publication title -
journal of molecular recognition
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.401
H-Index - 79
eISSN - 1099-1352
pISSN - 0952-3499
DOI - 10.1002/jmr.749
Subject(s) - cohesin , cellulosome , tandem affinity purification , fusion protein , biochemistry , biology , chemistry , protein–protein interaction , plasma protein binding , clostridium thermocellum , affinity chromatography , enzyme , recombinant dna , dna , chromatin , cellulase , gene
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. Copyright © 2005 John Wiley & Sons, Ltd.