Answer to Question #153641 in Biochemistry for Mani

Question

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.

Answer 1

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.

References

(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,