title: Cloning and deletion mutagenesis using direct protein-protein interaction on an expression vector. Identification of the calmodulin binding domain of alpha-fodrin.
abstract: We have screened a lambda gt11 library, constructed with mouse macrophage cDNA, in order to isolate clones that code for calmodulin binding proteins. We have developed a new approach for this purpose using radioactive calmodulin (produced by genetic engineering) to detect fusion proteins that interact with this protein with high affinity. A cDNA clone that codes for mouse macrophage fodrin was isolated, sequenced and identified. By deleting part of the sequence the calmodulin binding domain was located on the fodrin sequence. The site is situated on repeat 11 of fodrin and probably on the extra arm of this repeat. The method we developed is widely applicable to site-directed mutagenesis of interacting proteins.
authors.affiliation: U-249 INSERM, CRBM du CNRS & Université, Montpellier, France.
authors.forename: J
authors.initials: J
authors.lastname: Sri Widada
authors.forename: J
authors.initials: J
authors.lastname: Asselin
authors.forename: S
authors.initials: S
authors.lastname: Colote
authors.forename: J
authors.initials: J
authors.lastname: Marti
authors.forename: C
authors.initials: C
authors.lastname: Ferraz
authors.forename: G
authors.initials: G
authors.lastname: Travé
authors.forename: J
authors.initials: J
authors.lastname: Haiech
authors.forename: J P
authors.initials: JP
authors.lastname: Liautard
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002147:Calmodulin / Q000378:metabolism*; D002352:Carrier Proteins / Q000378:metabolism*; D002872:Chromosome Deletion; D003001:Cloning, Molecular; D051379:Mice; D008840:Microfilament Proteins / Q000378:metabolism*; D008969:Molecular Sequence Data; D009154:Mutation; D013049:Spectrin
chemical_list: D002147:Calmodulin; D002352:Carrier Proteins; D008840:Microfilament Proteins; D013049:Spectrin