Measurements of relative binding of cohesin and dockerin mutants using an advanced ELISA technique for high-affinity interactions

Michal Slutzki*, Yoav Barak, Dan Reshef, Ora Schueler-Furman, Raphael Lamed, Edward A. Bayer

*Corresponding author for this work

Research output: Chapter in Book/Report/Conference proceedingChapterpeer-review

5 Scopus citations

Abstract

The cellulosome is a large bacterial extracellular multienzyme complex able to degrade crystalline cellulosic substrates. The complex contains catalytic and noncatalytic subunits, interconnected by high-affinity cohesin-dockerin interactions. In this chapter, we introduce an optimized method for comparative binding among different cohesins or cohesin mutants to the dockerin partner. This assay offers advantages over other methods (such as ELISA, cELIA, SPR, and ITC) for particularly high-affinity binding interactions. In this approach, the high-affinity interaction of interest occurs in the liquid phase during the equilibrated binding step, whereas the interaction with the immobilized phase is used only for detection of the unbound dockerins that remain in the solution phase. Once equilibrium conditions are reached, the change in free energy of binding (ΔΔG binding ), as well as the affinity constant of mutants, can be estimated against the known affinity constant of the wild-type interaction. In light of the above, we propose this method as a preferred alternative for the relative quantification of high-affinity protein interactions.

Original languageAmerican English
Title of host publicationMethods in Enzymology
PublisherAcademic Press Inc.
Pages417-428
Number of pages12
DOIs
StatePublished - 2012

Publication series

NameMethods in Enzymology
Volume510
ISSN (Print)0076-6879
ISSN (Electronic)1557-7988

Keywords

  • Cohesin-dockerin interaction
  • High-affinity binding
  • Indirect ELISA

Fingerprint

Dive into the research topics of 'Measurements of relative binding of cohesin and dockerin mutants using an advanced ELISA technique for high-affinity interactions'. Together they form a unique fingerprint.

Cite this