Affinity-based chromatography utilizing genetically engineered proteins. Interaction of Bordetella pertussis adenylate cyclase with calmodulin.
An engineered calmodulin differs from vertebrate calmodulin in its ability to activate Bordetella pertussis adenylate cyclase, and this difference has been utilized as the basis for a new purification protocol for the adenylate cyclase. VU-8 calmodulin, in which 3 glutamic acid residues (residues 82-84) have been substituted with 3 lysine residues, has a 1000-fold lower apparent affinity for the adenylate cyclase, compared to vertebrate calmodulin, and decreased maximal activity. Because of the relatively calcium-independent nature of the interaction between calmodulin and the cyclase, the use of calmodulin-Sepharose conjugates in the purification of the cyclase requires the use of chaotropic agents for elution. However, when immobilized VU-8 calmodulin was tested as a calcium-dependent, affinity-based, adsorption chromatography step in the purification of the cyclase from culture media or bacterial extracts, the enzyme bound to the column in a calcium-dependent manner, and a nearly homogeneous enzyme was obtained in high yield. These results demonstrate the feasibility of using engineered calmodulins that have selective differences in activity for the rational design of rapid purification protocols for calmodulin-binding proteins as well as indicate the importance of the conserved negative charge cluster at residues 82-84 of calmodulin for activation of this cyclase.
D000262:Adenylyl Cyclases / Q000302:isolation & purification / Q000378:metabolism*; D000818:Animals; D001886:Bordetella pertussis / Q000201:enzymology*; D002147:Calmodulin / Q000235:genetics* / Q000378:metabolism; D002417:Cattle; D002846:Chromatography, Affinity; D005818:Genetic Engineering; D008722:Methods
D002147:Calmodulin; D000262:Adenylyl Cyclases