The production of recombinant calpastatin in E. coli has become an efficient tool to obtain discrete amounts of a specific calpastatin species that can be present concomitantly with other calpastatin fragments/forms in the same tissue or cell type in a given condition. Indeed, at present, it is still difficult to distinguish the various calpastatin species for several reasons among which: calpastatins differ only at the N-terminus, can undergo calpain-dependent cleavage generating discrete fragments, and show anomalous electrophoretic mobility. Another benefit of using recombinant calpastatin is that, as the wild-type forms, it is heat resistant and thus can be efficiently isolated taking advantage of a simple quick purification step. Finally, the lack of posttranslational modifications makes recombinant calpastatin species particularly suitable for studying in vitro the biochemical features of specific parts of the inhibitor that following controlled posttranslational modifications change their functional interaction with calpain. In this chapter, we describe, starting from the mRNA sequence, how to produce rat calpastatin Type I in E. coli. We use routinely the same method, with minor modifications, for the production of other calpastatin species deriving from different tissues or organisms and calpastatin constructs having only specific domains. The possibility to obtain large amounts of a single calpain inhibitor form is a great advantage for studying the calpain/calpastatin system in vitro.
This is a preview of subscription content, log in to check access.
Springer Nature is developing a new tool to find and evaluate Protocols. Learn more
This work was supported by the grant FRA2015 and FRA2016 from University of Genova to MA and RDT.
Takano J, Watanabe M, Hitomi K, Maki M (2000) Four types of calpastatin isoforms with distinct amino-terminal sequences are specified by alternative first exons and differentially expressed in mouse tissues. J Biochem 128:83–92CrossRefGoogle Scholar
Parr T, Sensky PL, Bardsley RG, Buttery PJ (2001) Calpastatin expression in porcine cardiac and skeletal muscle and partial gene structure. Arch Biochem Biophys 395:1–13CrossRefGoogle Scholar
Lee WJ, Ma H, Takano E, Yang HQ, Hatanaka M, Maki M (1992) Molecular diversity in amino-terminal domains of human calpastatin by exon skipping. J Biol Chem 267:8437–8442PubMedGoogle Scholar
De Tullio R, Sparatore B, Salamino F, Melloni E, Pontremoli S (1998) Rat brain contains multiple mRNAs for calpastatin. FEBS Lett 422:113–117CrossRefGoogle Scholar
Takano J, Kawamura T, Murase M, Hitomi K, Maki M (1999) Structure of mouse calpastatin isoforms: implications of species-common and species-specific alternative splicing. Biochem Biophys Res Commun 260:339–345CrossRefGoogle Scholar
De Tullio R, Averna M, Stifanese R, Parr T, Bardsley RG, Pontremoli S, Melloni E (2007) Multiple rat brain calpastatin forms are produced by distinct starting points and alternative splicing of the N-terminal exons. Arch Biochem Biophys 465:148–156CrossRefGoogle Scholar
De Tullio R, Averna M, Salamino F, Pontremoli S, Melloni E (2000) Differential degradation of calpastatin by mu- and m-calpain in Ca2+-enriched human neuroblastoma LAN-5 cells. FEBS Lett 475:17–21CrossRefGoogle Scholar
Averna M, De Tullio R, Passalacqua M, Salamino F, Pontremoli S, Melloni E (2001) Changes in intracellular calpastatin localization are mediated by reversible phosphorylation. Biochem J 354:25–30CrossRefGoogle Scholar
De Tullio R, Cantoni C, Broggio C, Prato C, Stifanese R, Averna M, Antolini R, Pontremoli S, Melloni E (2009) Involvement of exon 6-mediated calpastatin intracellular movements in the modulation of calpain activation. Biochim Biophys Acta 1790:182–187CrossRefGoogle Scholar
Geesink GH, Nonneman D, Koohmaraie M (1998) An improved purification protocol for heart and skeletal muscle calpastatin reveals two isoforms resulting from alternative splicing. Arch Biochem Biophys 356:19–24CrossRefGoogle Scholar
Parr T, Sensky MK, Bardsley RG, Buttery PJ (2000) Effects of epinephrine infusion on expression of calpastatin in porcine cardiac and skeletal muscle. Arch Biochem Biophys 374:299–305CrossRefGoogle Scholar
Vincze T, Posfai J, Roberts RJ (2003) NEBcutter: a program to cleave DNA with restriction enzymes. Nucleic Acids Res 31:3688–3691CrossRefGoogle Scholar