Question

a-d plz

7. Protein purification. As a graduate student, the first protein I purified was RNA polymerase (RNAP) from E. coli- Some physical and chemical properties of Ecoli RNAP Molecular max470,000 g/mol polynentide compasitian (subunits): (50 kDa), k andok a pl = 5.34 substrates: NTPs cofactor: Mg? Purification protocol. E coli cells were broken usine Isozyme, yielding a cellualar extract containing a protcome solution 4M (NHOSO, was added to the cellular extract. A white protein precipitate was formed incuding RNAM The protein precipitate was spun down in a centrifuge and dialyzed into an aqueous buffer of 10 mM TripH 7.4. A) The proteome solution was fractionated by ion-exchange chromatography. What type of ion exchange resin will bind RNAP at pH 7.4? 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 D) 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 470 kDa. However, when I analyzed the pure RNAP by SDS- PAGE, I observed not one but three bands corresponding to separated polypeptides with masses of 50, 70, and 150 kDa. When I removed the B-mercaptoethanol from the SDS-PAGE, 1 observed three bands with masses of 70, 100, and 300. Rationalize these contrasting data.

Answer 1

A) The resin bed used for the purpose would be an anion-exchanger (NR⁺ OH^-)to bind to RNAP at pH 7.4

B) To elute the RNAP from the column NaCl would have been used since the Cl^- ions would themselves bind to the anion-exchange bed releasing the RNAP which flows down the column and gets eluted.

C) For the affinity chromatography the suitable ligand to bind to RNAP is epoxide, which is usually generated by treating the anion-exchange bed with epichlorohydrin that reacts with hydroxyl (OH^-) groups to form epoxide.

D) B-Mercaptoethanol is used for breaking disulfide bridges in the protein thereby denaturing it. The unfolded protein hence migrates in such state and we get three bands corresponding to the molecular weights of three subunits. On excluding the use of B-mercaptoethanol the protein rather is in its native folded form and migrates as such, thus, presenting three bands which do not correspond to actual molecular mass of the subunits but when added gives the actual molecular mass of the protein as a whole.