Production of Reagents and Optimization of Methods for Studying Calmodulin-Binding Proteins

• Published in: Protein expression and purification Vol. 15; no. 1; pp. 24 - 33
• Main Authors: Ulbricht, Bettina, Soldati, Thierry
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 01.02.1999
• Subjects: affinity chromatography; Animals; Blotting, Western; calmodulin; Calmodulin - genetics; Calmodulin - isolation & purification; Calmodulin - metabolism; calmodulin-binding proteins; Calmodulin-Binding Proteins - metabolism; Chromatography, Affinity; Chromatography, Ion Exchange; Cloning, Molecular - methods; Cytosol - metabolism; Dictyostelium - genetics; Dictyostelium - metabolism; Dictyostelium discoideum; Electrophoresis, Polyacrylamide Gel; Escherichia coli; Fluorescent Antibody Technique; Indicators and Reagents; myosins; Recombinant Proteins - biosynthesis; Recombinant Proteins - isolation & purification; Recombinant Proteins - metabolism; Dictyostelium discoideum; calmodulin-binding proteins; affinity chromatography; calmodulin; myosins
• ISSN: 1046-5928; 1096-0279
• DOI: 10.1006/prep.1998.0983

Owing to subtle but potentially crucial structural and functional differences between calmodulin (CaM) of different species, the biochemical study of low-affinity CaM-binding proteins fromDictyostelium discoideumlikely necessitates the use of CaM from the same organism. In addition, most of the methods used for identification and purification of CaM-binding proteins require native CaM in nonlimiting biochemical quantities. The gene encodingD. discoideumCaM has previously been cloned allowing production of recombinant protein. The present study describes the expression ofD. discoideumCaM inEscherichia coliand its straightforward and rapid purification. Furthermore, we describe the optimization of a complete palette of assays to detect as little as nanogram quantities of proteins binding CaM with middle to low affinities. Purified CaM was used to raise high-affinity polyclonal antibodies suitable for immunoblotting, immunofluorescence, and immunoprecipitation experiments. The purified CaM was also used to optimize a specific and sensitive nonradioactive CaM overlay assay as well as to produce a high-capacity CaM affinity chromatography matrix. The effectiveness of this methods is illustrated by the detection of potentially novelD. discoideumCaM-binding proteins and the preparatory purification of one of these proteins, a short tail myosin I.