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Yield was 50–75 mg of Ca M per 500 ml of bacterial culture, and Ca M was homogeneous as judged by SDS-polyacrylamide gel electrophoresis.
For purification, cell pellets were resuspended in 50 m EGTA, and protein was detected by the Bradford method.
Ca M was detected by the Bradford method (Bio-Rad), and the appropriate fractions were pooled.
In the second method of Ca M purification, cell pellets that had been resuspended in lysis buffer (50 m MOPS, p H 7.0 (ultra-free spin column, molecular weight 5000 cutoff, Millipore).
All mutagenesis was performed with the Transformer site-directed mutagenesis kit (CLONTECH), and all mutations were verified by DNA sequencing.
Ca M was expressed in the bacterial strain BL21(DE3) (Novagen).