Question

You are to purify a highly soluble homotetrameric protein containing a NADP binding site, in its functional form, and a pI = 9 with a subunit molecular weight of 35,000 Da from a crude extract of E. coli. For this protein, select the four best techniques from the list below and place them in the correct order for the most reasonable strategy to obtain homogeneous protein.

(A) Precipitation of proteins using high ionic strength (40%) ammonium sulfate, followed by a second precipitation with 60% (w/v) ammonium sulfate in a batchwise precipitation.

(B) SDS-PAGE for 0.01 mg total protein

(C) Ultracentrifugation in a sucrose density gradient

(D) Cation exchange with CM-Sepharose at pH 7.5

(E) Anion exchange with DEAE-Sepharose at pH 7.5

(F) Affinity chromatography with Cibacron Blue-Sepharose (binds to NAD/NADP binding sites)

(G) Gel filtration with Sephadex, cation exchange chromatography with CM-Sepharose.

(H) Non-denaturing gel electrophoresis for 0.01 mg total protein

Answer 1

1. Firstly, we solubilize the proteins from the crude extract by salting and then precipitate by salting out. For this we, use (A) precipitation of protein using 40% strength ammonium sulfate, because at lower salt concentration solubility of proteins increase with increase in salt concentration. So, by this process we solubilize the proteins from crude extract, after that increase the salt concentration to 60% due to this desired protein precipitate( at high salt concentration interaction between water and protein decreases and protein protein interaction increases which cause formation of ppt of protein).

2. In question it is given at PI of protein is 9, so at pH below 9 it acquire positive charge. Therefore (D) Cation exchange with CM-Sepharose at pH 7.5 is used. So, the all the protein that have PI more than 7.5 or have positive charge obtained.

3. It is given that sub unit molecular weight is 35,000 Da and protein of interest is homo tetrameric. So the total weight of protein is 140,000 Da. Separation of protein by molecular weight can be done by PAGE but non-denaturing, because it is given that protein contain NADP binding site at function form and we need the protein in function form for the last step. That's why we use (H) non-denaturing gel electrophoresis( SDS PAGE denature the protein or we can say protein lost its functional form) for separation.

4. It is given that NADP binding site is present in functional form. Therefore, we can use (F) affinity chromatography with Cibacron Blue-Sepharose( binds to NAD/NADP binding site). After this step pure protein of interest is obtained .