Matching fusion protein systems for affinity analysis of two interacting families of proteins: the cohesin-dockerin interaction.
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.
Barak
Yoav
Y
Department of Biological Chemistry, The Weizmann Institute of Science, Rehovot 76100, Israel.
Handelsman
Tal
T
Nakar
David
D
Mechaly
Adva
A
Lamed
Raphael
R
Shoham
Yuval
Y
Bayer
Edward A
EA
D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D018797:Cell Cycle Proteins / Q000235:genetics / Q000378:metabolism*; D002868:Chromosomal Proteins, Non-Histone; D004797:Enzyme-Linked Immunosorbent Assay; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism*; D009097:Multienzyme Complexes; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism*; D010940:Plant Proteins / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism*; D015203:Reproducibility of Results; D014995:Xylosidases / Q000235:genetics / Q000378:metabolism; D000097722:Cohesins
D001426:Bacterial Proteins; D018797:Cell Cycle Proteins; D002868:Chromosomal Proteins, Non-Histone; D005656:Fungal Proteins; D009097:Multienzyme Complexes; D009687:Nuclear Proteins; D010940:Plant Proteins; D011993:Recombinant Fusion Proteins; D014995:Xylosidases; D000097722:Cohesins