experiment 4: purification of rGFP using Ni2+ agarose

Question 1

what is the underlying principle that allows for the purification of rGFP?

Answer 1

• rGFP contains a His-6 tag, which binds to the Ni 2+ agarose in the affinity column
• after the sample is applied to the column, the column is washed with breaking buffer, which removes any proteins that aren’t rGFP (because they do not contain a His-6 tag and therefore do not bind to the column)
• then the column is eluted with elution buffer, which contains imidazole. imidazole has a very similar structure to histidine and a higher affinity for the column. this causes displacement of the His-6 tagged rGFP, which is collected in the elution samples

Question 2

why does freezing and then thawing the bacterial pellet result in lysis?

Answer 2

• as the cells are slowly frozen, ice crystals form within the cell in the cytoplasm and in the cell walls, which can cause these walls to become punctured
• the cells are then quickly thawed, and the few cells whose walls were damaged burst open due to the osmotic shock
• they spill their cytoplasm, which releases lysozyme that degrades the neighboring cell walls and a chain reaction of lysing begins
• note: this process is not 100% efficient: some cells remain unbroken, so the rGFP remained within the cell
• the thawing process takes place in a water bath near the optimal temperature for lysozyme activity which leads to more efficient breaking

Question 3

what is the purpose of the “wash” step when developing the column?

Answer 3

• the wash step washes off any unbound proteins. rGFP is the only protein that contains the His-6 tag, so should be the only protein that binds to the column
• it can be concluded that any proteins that do not bind to the column are not the desired GFP
• note: if column is overloaded, there are no more binding sites for His-6 tag, and some of the rGFP is washed off; this reduces yield in elutions

Question 4

how are we monitoring the presence of rGFP?

Answer 4

• qualitatively by observing fluorescence under a UV lamp
• quantitatively by measuring RFUs with a spectrofluorometer

Question 5

if we were purifying a histidine tagged phosphatase, how would we monitor its presence during the purification procedure?

Answer 5

• phosphatase is an enzyme, so we would have to perform an enzyme assay to detect its presence
• purifying a histidine tagged phosphatase would require a procedure similar to that in Experiment 2, where the phosphatase was added to a tube containing a substrate, such a phenolphthalein-diphosphate
• the enzyme would dephosphorylate the substrate and after 30 minutes, sodium carbonate would be added to stop the reaction
• the presence of histidine tagged phosphatase would be indicated by the intensity of the resulted pink color of the solution

Question 6

what is the underlying principle that allows for the elution of rGFP from the Ni2+ agarose column?

Answer 6

• the elution buffer contains the compound imidazole, which has a very similar structure to histidine
• because the Ni-agarose column has a greater affinity to the imidazole than it does with the His6 tag, the imidazole in the elution buffer competes with the rGFP for binding
• in the end, the greater affinity and larger amount of imidazole causes the rGFP to be eluted out of the column

Question 7

what are the five major types of column chromatography and the underlying principles that make them useful for separating proteins?

Answer 7

• gel-filtration chromatography: separates according to molecular weight
• ion-exchange chromatography: separates according to charge. includes anion-exchange which binds negative charge (so column is positively charged) and cation-exchange which binds positive charge (column is negatively charged)
• hydrophobic interaction chromatography: oily columns; anything that’s hydrophobic will want to stick to the hydrophobic residues and can elude off by changing pH (not used very often)
• affinity chromatography: separates according to specific molecular binding properties (for example in Lab 4, binding properties were based on shape of binding site and substrate shapes (histidine vs imidazole))

NOTE: Gel filtration, ion-exchange, and affinity chromatography are most common