Abstract
The production of recombinant proteins has been a cornerstone of the study of protein structure and function. As an example, the expression and purification of recombinant rat calpain-2 in bacteria was essential for solving the first crystal structures of the calpains in both calcium-free and calcium-bound forms. Here we describe the production and purification of recombinant rat calpain-2 from Escherichia coli using anion-exchange, affinity, and size-exclusion chromatographies. The heterodimeric enzyme is produced from a stable two-plasmid system. The order in which the protocol is carried out has been optimized to reduce unnecessary concentration and dialysis steps. The typical yield of this multi-domain enzyme from 4 L of E. coli culture is about 20 mg. The production of whole structures for the other calpain family members has been fraught with difficulty. To circumvent this roadblock, a certain amount of structure-function information can be gleaned about these other calpain isoforms by expressing just their protease core. These “mini-calpains” have been useful for X-ray co-crystallography with calpain inhibitors.
Here we also present a variation of the whole enzyme production and purification protocol optimized for the expression and purification of the calpain-1 and calpain-3 protease cores (mini-calpains).
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References
Campbell RL, Davies PL (2012) Structure-function relationships in calpains. Biochem J 447(3):335–351. https://doi.org/10.1042/BJ20120921
Goll DE, Thompson VF, Li H, Wei W, Cong J (2003) The calpain system. Physiol Rev 83(3):731–801. https://doi.org/10.1152/physrev.00029.2002
Elce JS, Hegadorn C, Gauthier S, Vince JW, Davies PL (1995) Recombinant calpain II: improved expression systems and production of a C105A active-site mutant for crystallography. Protein Eng 8(8):843–848
Chou JS, Impens F, Gevaert K, Davies PL (2011) m-Calpain activation in vitro does not require autolysis or subunit dissociation. Biochim Biophys Acta 1814(7):864–872. https://doi.org/10.1016/j.bbapap.2011.04.007
Moldoveanu T, Hosfield CM, Lim D, Elce JS, Jia Z, Davies PL (2002) A Ca(2+) switch aligns the active site of calpain. Cell 108(5):649–660
Sorimachi H, Toyama-Sorimachi N, Saido TC, Kawasaki H, Sugita H, Miyasaka M, Arahata K, Ishiura S, Suzuki K (1993) Muscle-specific calpain, p94, is degraded by autolysis immediately after translation, resulting in disappearance from muscle. J Biol Chem 268(14):10593–10605
Federici C, Eshdat Y, Richard I, Bertin B, Guillaume JL, Hattab M, Beckmann JS, Strosberg AD, Camoin L (1999) Purification and identification of two putative autolytic sites in human calpain 3 (p94) expressed in heterologous systems. Arch Biochem Biophys 363(2):237–245. https://doi.org/10.1006/abbi.1998.1091
Rey MA, Davies PL (2002) The protease core of the muscle-specific calpain, p94, undergoes Ca2+−dependent intramolecular autolysis. FEBS Lett 532(3):401–406
Diaz BG, Moldoveanu T, Kuiper MJ, Campbell RL, Davies PL (2004) Insertion sequence 1 of muscle-specific calpain, p94, acts as an internal propeptide. J Biol Chem 279(26):27656–27666. https://doi.org/10.1074/jbc.M313290200
Schagger H, von Jagow G (1987) Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis for the separation of proteins in the range from 1 to 100 kDa. Anal Biochem 166(2):368–379
Strobl S, Fernandez-Catalan C, Braun M, Huber R, Masumoto H, Nakagawa K, Irie A, Sorimachi H, Bourenkow G, Bartunik H, Suzuki K, Bode W (2000) The crystal structure of calcium-free human m-calpain suggests an electrostatic switch mechanism for activation by calcium. Proc Natl Acad Sci U S A 97(2):588–592
Acknowledgments
We thank Dr. Laurie Graham for her suggestion for monitoring the success of sonication using the Bradford assay. This work was funded by the Canadian Institute of Health Research. PLD holds a Canada Research Chair in Protein Engineering.
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McCartney, CS.E., Davies, P.L. (2019). Bacterial Expression and Purification of Calpains. In: Messer, J. (eds) Calpain. Methods in Molecular Biology, vol 1915. Springer, New York, NY. https://doi.org/10.1007/978-1-4939-8988-1_2
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DOI: https://doi.org/10.1007/978-1-4939-8988-1_2
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