Abstract
The use of skeletal muscle troponin C (TnC) in structural and functional studies ranging from characterization of its Ca2+-binding properties to interaction with troponin I (TnI) and troponin T (TnT), the other two members of the tripartite troponin regulatory complex, has necessitated the purification of large quantities of protein. The procedures for purification of the naturally occurring troponin components from muscle tissue, including preparation of a muscle ether powder, isolation of the crude troponin complex, and further fractionation using column chromatography to obtain the individual subunits, have been described in detail by Potter (1), and therefore, will not be discussed in this chapter. Owing to the problems with heterogeneity arising from copurification of different isoforms and/or variation in the extent of phosphorylation of the final isolated protein from muscle tissue, methodologies have since been developed using molecular biology strategies to express and purify homogeneous preparations of TnC, TnI, and TnT. Other advantages of these recombinant techniques include the ability of producing site-specific mutants and isolated fragments for studying specific regions without interference from the rest of the molecule, as well as 15N- and 13C-labeled derivatives for NMR studies.
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References
Potter, J. D. (1982) Preparation of troponin and its subunits. Methods Enzymol. 85, 241–263.
Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) Bacterial media, antibiotics,and bacterial strains, in Molecular Cloning: A Laboratory Manual, vol. 3, ColdSpring Harbor Laboratory Press, Cold Spring Harbor, NY, pp A.1–A.6.
Xu, G.-Q. and Hitchcock-DeGregori, S. E. (1988) Synthesis of aTroponin C cDNAand expression of Wild-type and mutant proteins in Escherichia coli. J. Biol. Chem. 263, 13,962–13,970.
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© 2002 Humana Press Inc.
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Pearlstone, J.R., Smillie, L.B. (2002). Skeletal Muscle Troponin C. In: Vogel, H.J. (eds) Calcium-Binding Protein Protocols. Methods in Molecular Biology™, vol 172. Humana Press. https://doi.org/10.1385/1-59259-183-3:161
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DOI: https://doi.org/10.1385/1-59259-183-3:161
Publisher Name: Humana Press
Print ISBN: 978-0-89603-688-8
Online ISBN: 978-1-59259-183-1
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