8. Protein purification. As a graduate student, the first protein I purified was RNA polymerase (RNAP) from E. coli. Some physical and chemical properties of E. coll RNAP Molecularmass =470,000g/mol polypeptide composition (subunits): (50kDa),(150kDa) and (70kDa) pl =5.34 substrates: NTPs cofactor: Mg2+ Purification protocol. E, coll cells were broken using lysozyme, yielding a cellualar extract containing a proteome solution. 4M(NH4)2SO4 was added to the cellular extract. A white protein precipitate was formed (including RNAP). The protein precipitate was spun down in a centrifuge and dialyzed into an aqueous buffer of 10mM Tris pH7.4. A) The proteome solution was fractionated by ion-exchange chromatography. What type of ion-exchange resin will bind RNAP at pH 7.4? AniUn exchanger (W R ROO ? B) How was RNAP eluted from this column? Explain why. C) RNAP was further purified by affinity chromatography. What is an excellent ligand to attach to an inert resin, which will bind RNAP with high affinity and specificity? [Think about a specific DNA sequence.] Finally, RNAP was purified to homogenity by gel-filtration chromatography. The RNAP eluted from the column as a single peak with apparent molecular mass of 470kDa. However, when I analyzed the pure RNAP by SDSPAGE, I observed not one but three bands corresponding to separated polypeptides with masses of 50 , 70 , and 150kDa. When I removed the -mercaptoethanol from the SDS-PAGE, I observed three bands with masses of 70 . 100, and 300 . Rationalize these contrasting data