Calpain 1 is a cysteine-protease existing as a heterodimer made up of a large and small subunit. The large subunit is 714 residues long (mass 81.9 kDa) with a pI of 5.49. The small subunit is 268 residues long (mass 28.3 kDa) with a pI of 5.05. Calpain 1 is inhibited by calpastatin, an intrinsically disordered protein of 708 residues (mass 76.5 kDa) with a pI of 4.97. Suggest a purification method that would allow you to separate calpain 1 from calpastatin. Describe conditions and explain why the chosen method would work and give reasons why it wouldn’t work.
Calpains forms one of the largest group of proteases that are vital in the body as they cleave various proteins responsible for signal transduction cascades. Calpain-1 specifically is known to be activated by calcium-binding in order to function in the vascular smooth muscle where it is responsible for remodeling the extracellular matrix of vascular walls. (2021) This occurs through crosslinking of calpain-1 to MMP-2 protein hence increasing transcription and translation of collagen. Calpastatin on the other hand is an endogenous inhibitor of calpain- 1.
Separation of the two proteins has been traditionally done using complex chromatographic methods which unfortunately has proven tedious and time-consuming. The most recent and effective way of separating calpain- 1 from calpastatin is the affinity purification method. The principle of this technique lies in the property of calpain-1 binding reversibly to the altered protein, calpastatin, in calcium-mediated reaction. (Nguyen, Varadi, Tompa & Pauwels, 2021) Calpastatin has a binding domain for calcium known as hcsd- 1 which has a high affinity for calcium. The dissociation of the resulting complex is accomplished through chelating calcium leading to elution of the desired calpain-1 and leaving the hcsd-1 in an immobile and stationary phase. (Nguyen, Varadi, Tompa & Pauwels, 2021) Compared to other conventional methods used in the purification of calpain -1, the method generates a high and robust yield of calpain which can, in turn, be subjected to further studies. The technique also maintains the required biophysical condition of the protein, calpain-1.
(2021). Retrieved 4 January 2021, from https://www.researchgate.net/publication/301776372_Chapter_13_The_Aging_Arterial_Wall
Nguyen, H., Varadi, M., Tompa, P., & Pauwels, K. (2021). Affinity purification of human m-calpain through an intrinsically disordered inhibitor, calpastatin. Retrieved 4 January 2021,