Question

You are assigned to purify a protein from an E.coli cell lysate. The following information is provided to you about the target protein.

1. The protein has an N-terminal 6X histidine tag (His-tag) attached to it

2. The protein is very stable in PBS (Phosphate Buffered Saline) buffer that you need to use as the storage buffer after purification

3. It is an enzyme that will be used as a potential drug.

4. You have the amino acid sequence of the protein.

Based on the information above, Answer the following questions:

1. How would you purify the protein from the lysate? Explain in detail your rationale for choosing the specific technique (3 marks)

2. How would you confirm the presence and purity of your protein after purification step? Do you think the sequence of the protein will help you to find some necessary information for this step? (3 marks)

3. What will be the best way to change the buffer after the purification? Briefly explain. (2 marks)

4. As a drug target, what should be the most important property of your target protein that is needed to be checked before proceeding to large scale production? (2 marks)

(Hint: It is an enzyme)

Answer 1

I. Ultracentrifugation can be used to purify proteins from the cell lysate.Since the amino acids sequence is known the approximate density can be calculated and the purification based on density difference is a reliable method.

II.Techniques like immunoprecipitation of the protein can be used.This not only detects the presence the protein but also purity of the protein can be assesed by the degree of precipitation.(In this technique, an antibody binds to the specific protein ,in this case the enzyme)

Alternate method :ELISA

III.This can be done by dializing the sample into a bulk amount of the new buffer.

IV.The enzyme should display great affinity towards the drug target with a high degree of specificity.