Indirect ELISA-based approach for comparative measurement of high-affinity cohesin-dockerin interactions.

摘要: The interaction between the cohesin and dockerin modules serves to attach cellulolytic enzymes (carrying dockerins) non-catalytic scaffoldin units multiple cohesins) in cellulosome, a multienzyme plant cell-wall degrading complex. This is species-specific, for example, enzyme-borne from Clostridium thermocellum bacteria binds cohesins same but not cellulolyticum vice versa. We studied role of interface residues, contributing either affinity or specificity, by mutating these residues on counterpart C. thermocellum. high cognate interactions makes it difficult evaluate effect mutations common methods used measuring protein-protein interactions, especially when subtle discrimination mutants needed. described this article an approach based indirect enzyme-linked immunosorbent assay (ELISA) that able detect differences binding various mutants, whereas surface plasmon resonance standard ELISA failed distinguish high-affinity interactions. To be calculate changes energy (ΔΔG) dissociation constants (K(d)) relative wild type, pre-equilibrium step was added procedure. Thus, cohesin-dockerin under investigation occurs solution rather than soluble immobilized proteins. Unbound dockerins are then detected through their with cohesins. Because our method allows us assess particularly tenacious much more accurately do other prevalent measure affinity, we therefore suggest as choice comparing