Expression of human calmodulin cDNA in Escherichia coli and characterization of the protein.
A cDNA clone of human calmodulin, isolated from liver, was subcloned into the expression vector pKK233-2. The resulting expression plasmid, designated pCWCaM1, produced human calmodulin in Escherichia coli SG5. The cDNA was sequenced using novel primers designed for use in plasmid-sequencing protocols with pKK233-2 and pKK223-3. The expressed calmodulin was purified and subjected to NMR analysis which revealed a structure essentially the same as natural calmodulin isolated from human tissue. The activation of myosin light chain kinase by the genetically engineered human calmodulin and bovine brain calmodulin was studied and found to be comparable to a high degree. The expressed calmodulin appears to be comparable to normal calmodulin and can be used for site-directed mutagenesis and structure/function investigations.
West
C A
CA
Cranfield Institute of Technology, Biotechnology Centre, Bedford, UK.
Bannister
J V
JV
Levine
B A
BA
Perham
R N
RN
D001483:Base Sequence; D002147:Calmodulin / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D004247:DNA / Q000032:analysis; D004926:Escherichia coli / Q000235:genetics; D005818:Genetic Engineering; D005822:Genetic Vectors; D006801:Humans; D008099:Liver / Q000032:analysis; D009682:Magnetic Resonance Spectroscopy; D009219:Myosin-Light-Chain Kinase / Q000032:analysis; D010957:Plasmids; D011994:Recombinant Proteins / Q000096:biosynthesis
D002147:Calmodulin; D011994:Recombinant Proteins; D004247:DNA; D009219:Myosin-Light-Chain Kinase