Matching fusion protein systems for affinity analysis of two interacting families of proteins: The cohesin-dockerin interaction

Yoav Barak, Tal Handelsman, David Nakar, Adva Mechaly, Raphael Lamed, Yuval Shoham, Edward A. Bayer

Research output: Contribution to journalArticlepeer-review

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.

Original languageEnglish
Pages (from-to)491-501
Number of pages11
JournalJournal of Molecular Recognition
Volume18
Issue number6
DOIs
StatePublished - Nov 2005

Keywords

  • Cellulosome
  • ELISA
  • Modular proteins
  • Multi-protein complex
  • Protein families
  • Protein-protein interactions

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