Question

Using the yeast cells to produce a small, negatively charged protein, explain the steps in purifying this protein.

For many steps there are a few correct answers but explain using a size exclusion column followed by an ion exchange column. (8 points)

Answer 1

First, we need to use size exclusion chromatography. Size exclusion chromatography works in the principle of the size of the protein. Bigger molecules are eluted first the smaller molecules. In this chromatography, we use the porous beads whose pore size depends on the size of the molecules. If the protein passes through the pore then it is eluted in the last.

Suppose our protein has a size of 10Kda. We need to use the beads whose cutoff size is 10 kDa. Proteins which are around 10Kda and smaller than 10kDa will pass through the bead pore whereas protein which is larger than 10kDa will not be able to pass the bead pore. So larger proteins will be eluted first then proteins whose size is 10Kda and smaller than 10kDa

To separate protein which is negatively charged, we need to use Ion-exchange chromatography. Since protein has an overall negative charge we will use cation exchange beads which has a positive charge such as diethyl-aminoethyl groups (DEAE).

The binding affinity of all the proteins with the beads differs due to their overall negative charge. After binding reaction when we pass the buffer whose pH is equal to the pI of the protein, Our protein will be eluted.

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