a) You have a solution containing five problems: Protein A, 48 kDa, p1 8.2 Prote
ID: 536344 • Letter: A
Question
a) You have a solution containing five problems: Protein A, 48 kDa, p1 8.2 Protein B, 60 kDa, p1 5.5 Protein C, 17 kDa, p1 5.7 Protein D, 18 kDa, p1 7.9 Protein E (MBP tagged), 58 kDa, p1 8.0 Using ONLY column chromatography, describe how you might purify Protein E from the original mixture containing ALL of the proteins listed above in a single purification step. Name the type of purification used and provide details - what type of column would be used and how is separation achieved? Your answer should include any required buffer components. b) What environmental (buffer) conditions are necessary to maximize protein interactions with the resin beads (solid phase) in hydrophobic interaction chromatography (HIC)? As part of your answer, clearly explain the protein - resin interactions and explain chemically how these interactions are maximized. Describe in DETAIL the hydrophobic effect as it relates to protein folding and structure. As part of your answer, you must explain what it is and clearly describe its role in protein folding and stability.Explanation / Answer
5.
a. Initially size-exclusion chromatography would separate protein C and D with relatively much lower molar masses from the remaining proteins A, B and E with higher molar masses.
Now A, B and E are separated by ion-exchange chromatography with anion exchange column with slowly increasing the pH and according to the pI of the proteins, protein E can be easily separated from the other two protein components.
b. Using bicarbonate buffer system would bring the hydrophobic groups of the system close to each other and hydrophobic interaction between groups are thus enhanced. Its like formation of micelle in a bocarbonate buffer system. The hydrophobic parts of the protein would stay close to the resin hydrophobic part which are long hydrocarbon chains and interact with one-another.
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