Question

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 E. coli RNAP Molecular mass = 470,000 g/mol polypeptide composition (subunits): a (50 kDa), B (150 kDa) and 6 (70 kDa) pl = 5.34 substrates: NTPS cofactor: Mg Purification protocol. E. coli 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 10 mM Tris pH 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, I observed three bands with masses of 70, 100, and 300. Rationalize these contrasting data.

Answer 1

Answer-

7) A) Ion exchange chromatography is applicable for the separation of charged molecules. In this technique, ionic solutes show reversible electrostatic interactions with charged stationary phase, which commonly consists of an insoluble matrix with covalently attached ions (ion exchanger). The solutes which are introduced in the column may be negatively charged, positively charged or neutral under particular condition.The strength of interaction depends on charge density. The greater the charge density, the stronger the interaction. pI of RNAP is 5.34. pH of the wxperimental condition is 7.4. When ph is greater than pI of a solute, the solute donates H+ ion and becomes negatively charged. So in this condition negatively charged RNAP will bind to positively charged ion exchanger.   Anion exchanger (basic ion exchanger) is used for anion separation in ion exchanger chromatography. Example of such anion exchanger is DEAE-cellulose (Diethylaminomethyl).

B) Elution of RNAP from the column- The bound RNAP can be eluted by altering the pH of the eluting buffer. In general, cationic buffers (such as Diethanolamine, Tris) are used in anion exchanger resin. To elutes molecules from anion exchanger resin, a decreasing pH gradient is chosen. Molecules will elute when the pH gradient reaches their pI, because at that particular condition RNAP will no longer carry a net negative charge that allows them to interact with the column resin.

C) Affinity chromatography is a technique enabling purification of biomolecules with respect to biological function ar individual chemical structure. The substance to be purified is specifically adsorbed to ligand, immobilized by a covalent bond to matrix. An ligand that can be attached to a chromatography matrix is one of the requirements for successful affinity purification. RNA polymerase of E. coli can binds to a specific DNA sequence of DNA, which is located at promoter site. -35 and -10 regions contain some specific nucleotides (TTGACA at -35 region; TATAAT at -10 region). RNAP binds to these sites to initiate transcription. So thiz nucleotide sequences can be used as ligand dor separation of RNAP in affinity chromatography.

D) RNAP is consisting of 5 subunits (2 alpha, 2 beta and 1 sigma) which are linked together thus forming holenzyme. These subunits are strongly attached to each other. In Gel filtration chromatography, the enzyme is separated as a whole because this enzyme is not denatured. In SDS-PAGE, SDS is a detergent which denatures the proteins. SDS denatures RNAP to 5 subunits ( 2 alpha subunits, each of 50kDa; 2 beta subunits, each with 150 kDa; and 1 sigma subunit with 70 kDa. So after gel electrophoresis 3 bands will be formed. a) band at 150 kDa (darker because 2 beta subunits are there), b) band at 70 kDa ( lighter) and c) band at 50 kDa (darker). Role of beta- mercaptoethanol is to break disulfide bonds in proteins. So, after removing beta mercaptoethanol 2 alpha subunits will not dissociate from each other, same thing is happened to beta subunits. alpha, beta and sigma subunits will dissociate from each others forming 2 alpha subunits together (50+50 =100 kDa), 2 beta subunits together (150+150 =300 kDa) and 1 sigma subunit with 70 kDa. Important thing is that 2 alpha subunits will be bound to each other and so are beta subunits. So after gel electrophoresis 3 bands will be seen a) 70kDa, b)100 kDa, c) 300 kDa.