To obtain a large amount of VU-4 calmodulin (115, Lys to Ile). VU-4 calmodulin gene with Ptac promoter (680 bp) was cloned into a plasmid, pUC18. Newly cloned pUC18/VU-4 was transferred into E. coli UT481. The plasmid isolated from cell extracts of th...
To obtain a large amount of VU-4 calmodulin (115, Lys to Ile). VU-4 calmodulin gene with Ptac promoter (680 bp) was cloned into a plasmid, pUC18. Newly cloned pUC18/VU-4 was transferred into E. coli UT481. The plasmid isolated from cell extracts of the transformed E. coli was analyzed by restriction mapping and yielded the expected restriction endonuclease map with BamHI and EcoRI. Calmodulin was detected by Western blot analysis using chemiluminescence protocol and the estimated expression level was 60 pmole calmodulin/mg protein. Calmodulin was purified from the E. coli cell extracts by Ca^(2+)-dependent chromatography on a phenyl-Sepharose column. The purified calmodulin was identified as a mutant VU-4 calmodulin by SDS-PAGE and methyltransferase substrate test.