Two proteins (X and Y) differ significantly in their pI values derived based on

Question

Two proteins (X and Y) differ significantly in their pI values derived based on their amino acid sequence. Protein X has a pI of 8 and protein Y has a pI of 4.

a) What is the functional definition of pI.

b) Based on the difference in pI, state which chromatography technique you would choose to attempt to purify protein X from a mixture of proteins X and Y and briefly explain why.

c) Describe the key features of a purification procedure you could use that would result in retention of protein X on the column followed by its elution. Include in your answer the sequence of steps, the buffer conditions (pH, salt
concentration) at the different stages of the procedure and the reasons you would choose these conditions.

d) Why might the experiment not work as you anticipate?

Explanation / Answer

a) The pH at which the protein has a net charge of zero.

b) Ion exchange chromatography because it separates proteins on the basis of net charge. At a specific pH the proteins will differ in their net charge.

c) At pH 6 protein X will have a net positive charge and protein Y will have a net negative charge. Cation exchange should be used as it will more reliably result in retention of X on the column which could then be eluted with an increasing gradient of NaCl. Y is unlikely to bind to the column at pH 6 as both Y and the media will be negatively charged.

d) The theoretical pI based on the number and type of charged amino acids in the sequence is not necessarily the same as the experimental pI. eg, some charges not on the surface are likely to have a smaller effect on the behaviour of the (folded) protein on the column.

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