Reference no: EM133229525
Lysozyme will be isolated by ion-exchange method, at conditions of pH = pI (see above for principles). Ion-exchange can be carried out by column-based ion exchange chromatography, or resin-based, like this practical. The ion-exchange resin is carboxy-methylcellulose (CMC), and maintains negative charges in solution. Proteins which have a net positive charge will bind to it, and those which have a net negative charge will not. The chicken egg-white lysozyme is a strongly basic protein of pI 9.32 (Worthington Enzyme Manual) (or pI 10.7, Abeyrathne et al., 2014; pI 11.3 Sigma Aldrich). Hence, in a solution of pH 9.0, lysozyme will have a net positive charge and bind to the CMC. In contrast, other proteins which have a pI lower than 9 (~80% of proteins) (e.g., ovalbumin, pI 4.5 (Sigma), or ovotransferrin, pI 6.0; Giansati et al., 2015) will be highly soluble at this pH, hence will not bind to the resin and can be washed away from it. In the next step, when the pH of the resin-washing solution is increased to 10.4, all proteins (including lysozyme) with pI close to 10.4 will have no net charge and will disassociate from the resin and can be collected. The proteins with pI above 10.4 will still remain bound, and can be eluted by a buffer of an even higher pH (or the resin can be discarded).
Fraction A (Crude egg white proteins)
Fraction B (CMC supernatant)
Fraction C (CMC pellet wash)
Fraction D (Eluted lysozyme)
Question 1. Of the 4 lysozyme fractions, which fraction did you expect to have the most lysozyme? Or least lysozyme? Why?
Question 2. How well did the purification of lysozyme with ion exchange work in terms of % recovery and % purity?
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