Draetta, G., Klee, C. B. (1988) Purification of Calmodulin-Stimulated Phosphodiesterase by Affinity-Chromatography on Calmodulin Fragment-1-77 Linked to Sepharose. Methods in Enzymology, 159. pp. 573-581. ISSN 0076-6879
Abstract
Because of the presence of many calmodulin-binding proteins in brain, affinity chromatography based on binding to calmodulin lacks specificity. Additional conventional purification steps are needed, prolonging the length of the purification procedure, causing a reduction in yield and increasing the probability of proteolysis and subsequent decrease of calmodulin stimulation. The lack of specificity of the calmodulin affinity chromatography step can be overcome by the use of calmodulin fragments. The N-terminal half-fragment of calmodulin, fragment 1-77, binds cyclic nucleotide phosphodiesterase but does not interact with most of the other calmodulin binding proteins present in brain extracts. Thus, affinity chromatogramaphy on fragment 1-77 coupled to Sepharose can be used to selectively purify the phosphodiesterase. A three-step method, based on the specific interaction of the enzyme with calmodulin 1-77, allows the rapid (2–3days) purification of cyclic nucleotide phosphodiesterase to a high specific activity and with retention of a large stimulation by calmodulin.
| Item Type: | Paper |
|---|---|
| Subjects: | biotechnology > chromatography > affinity chromatography organs, tissues, organelles, cell types and functions > organs types and functions > brain bioinformatics > genomics and proteomics > genetics & nucleic acid processing > protein structure, function, modification > protein types |
| CSHL Authors: | |
| Communities: | CSHL labs |
| Depositing User: | Gail Sherman |
| Date: | 1988 |
| Date Deposited: | 06 Oct 2017 15:48 |
| Last Modified: | 06 Oct 2017 15:48 |
| Related URLs: | |
| URI: | https://repository.cshl.edu/id/eprint/35138 |
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