You are trying to purify a new enzyme that is responsible for a key biosynthetic
ID: 533189 • Letter: Y
Question
You are trying to purify a new enzyme that is responsible for a key biosynthetic step in an important pathogenic bacterium so that you can determine its structure and develop a drug to inhibit its activity. The gene has been sequenced and shows a large number of lysine and arginine residues, which allow the protein to bind to another protein which has many aspartate and glutamate residues. You have been able to isolate the two proteins bound in a non-covalent complex but not each protein by itself. However if you dilute the solution of the two proteins, they readily dissociate.
Explain briefy how you might attempt to purify the protein of interest away from its binding partner using ion exchange chromatography, given only the information above. Provide details of the type of chromatography resin you would use.
What information would you need to know to decide on the pH at which to run your experiment? Why?
Sample question You are trying to purify a new enzyme that is responsible for a key biosynthetic step in an important pathogenic bacterium so that you can determine its structure and develop a drug to inhibit its activity. The gene has been sequenced and shows a large number of lysine and arginine residues, which allow the protein to bind to another protein which has many aspartate and glutamate residues. You have been able to isolate the two proteins bound in a non-covalent complex but not each protein by itself. However if you dilute the solution of the two proteins, they readily dissociate. Explain briefly how you might attempt to purify the protein ofinterest away from its binding partner using ion exchange chromatography, given only the information above. Provide details of the type of chromatography resin you 2 marks would use. What information would you need to know to decide on the pH at which to run your experiment? Why? 2 marksExplanation / Answer
As per the question the proteins contains many lysine and arginine residue. Both arginine and lysine can exist as cationic species Upton higher PH that means these have high affinity to hold the proton even if the PH is high.
Based on the above fact it's clear that ion exchange chromatography should be used.
The isoelectric point is responsible for the net charge of a protein in a particular PH. Accordingly exchange resin can be taken.
For the protein of our interest we can chose cation exchange resin and the resin will have negative charge and so it will bind with the positively charged protein due to ionic interaction. Due to different amount of interaction between molecules and resin those protein get separated and their elution time become different.
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