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KMID : 0578319940040010033
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Molecules and Cells
1994 Volume.4 No. 1 p.33 ~ p.37
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Expression Cloning Purification and Characterization of a Mutant Calmodulin
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Oh Suk-heung
Choi Dong-Seong
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Abstract
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In order to get higher expression of VU-3 calmodulin, VU-3 calmodulin gene fragment was cloned into a higher expression plasmid, pVUCH-1. Newly cloned pVUCH-3 plasmid was screened for the mutation by restriction mapping with BamH11HindIIl. In BamHI digestion, there was no difference between pVUCH-1 and pVUCH-3 plasmids. On the other hand, BamH1/HindIII digestion showed band shift due to the loss of the HindIIl site in the case of pVUCH-3. VU-3 calmodulin content in the extracts of bacterial clones containing pVUCH-3 was 33-fold higher than that in the extracts of bacterial clones carrying pVUC-3. VU-3 calmodulin was purified by Ca"-dependent chromatography on a phenyl-Sepharose column from the extracts of bacteria containing pVUCH-3. The purified VU-3 calmodulin was characterized by amino acid composition as well as methylation analysis. Overall, the pVUCH-3 system provides an effective way of producing VU-3 calmodulin in E. coll.
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