Question

4. Using only methods described in MCDB 310 and all the information above, devise a method to purify the DNA binding protein (NOT glycogen phosphorylase) from E. coli lysate (i.e. broken up bacteria in a buffer solution). You may only use two chromatography methods (not just two steps) for purification from the lysate, and you need the purest sample of the protein possible, so at least one method needs to be very specific. There is no antibody available.

Answer 1

Two chromatography methods for purification:

1. The first method to use will be cation exchange that includes separation by charge. In cation exchange, the protein will bind to the anionic resin. In this method positively charged particles or cations are attracted because in the cationic chromatography the resins are negatively charged and the proteins will bind if the buffer pH is less than the protein’s isoelectric point. The protein to be separated needs to be positively charged to bind to the negatively charged stationary phase.

2. The protein can be further purified by affinity chromatography using a DNA column to which the protein will bind.