Question

Protein purification. The protein tubulin assembles into microtubule fibers and is a major structural component of your neurons. Microtubule-associated proteins (“MAPs”) play a role in neuronal development by regulating the growth and morphology of microtubule structures. You are interested in studying the biochemical characteristics of a protein called “MAP2c”, which means that you need to purify it in an active form. Thus far, you have been able to able to isolate a mixture known as “microtubule protein”, which includes the following proteins:

a) the desired Map2c (positively charged at physiological pH, 70 kDa)

b) tubulin (negatively charged, 100 kDa)

c) Map2a (positively charged, 280 kDa)

d) Map2b (positively charged, 270 kDa)

9. Design a set of experiments to purify active MAP2c away from these contaminants (make sure that your procedure doesn't denature the protein). Explain how each step in your purification is expected to work and, by that. I mean after each step which proteins will still be in the mixture and which have you already separated out.

Answer 1

In the given case study protein purification mainly depends on two criteria i.e.,  Size and charge

Here we cannot follow SDS-PAGE as this method involves denatiration of protein by SDS.

First step: Purification based on charge

Ion-exchange chromatography can be used to seperate proteins based on net charge.

- Positively charged proteins (cationic proteins) can be separated on negatively charged carboxymethyl-cellulose (CM-cellulose) columns.

- Here positively charged proteins i.e., Map2c, Map2a, Map2b bind to a column of beads containing carboxylate groups, whereas a negatively charged protein will not.

-Positively charged proteins bound to the column can then be eluted by increasing the concentration of sodium chloride or another salt in the eluting buffer because sodium ions compete with positively charged groups on the protein for binding to the column.

Therefore at the end of first step, Map2c, Map2a, Map2b will remain in protein mixture.

Second step: Purification based on size:

Gel-filtration chromatography or Size exclusion chromatography is used to seperate out proteins based on size or molecular weight.

- The mixture is applied to the top of a column consisting of porous beads made of hydrated polymer such as dextran or agarose.

- In the given mixture, small protein molecules can enter these beads, but large ones cannot.The result is that small protein molecules i.e.,the desired Map2c (70 kDa) are distributed in the aqueous solution both inside the beads and between them, whereas large molecules Map2a (280 kDa) and Map2b (270 kDa) are located only in the solution between the beads.

- Large proteins flow more rapidly through this column and emerge first. Small molecules, which take a longer, tortuous path, will exit as last fraction. Therefore desired Map2c which is small among given mixture will elute out of column as last fraction (70 kDa).