A target protein was purified through several steps as shown in the table below.

Question

A target protein was purified through several steps as shown in the table below. Answer the following questions about this purification scheme as they relate to the "?" values within the table. What is the specific activity of the target protein after the first and fourth purification steps? Calculate the total protein yield (in milligrams) after the second and third purification steps. Calculate the overall percentage yield of the target protein (final activity/initial activity as a percentage) Consider five proteins with the properties shown in the following table. Answer the questions below about these proteins and justify your answers. Which protein would elute last from a gel filtration chromatography column under nondenaturing conditions? Give reasons. Which protein would migrate the slowest in an SDS-PAGE gel and why? Which protein would elute last from an anion exchange column using a buffer at 6.5? Rationalize your answer. What is the likely quaternary structure for protein D on the basis of the data in the table and why?

Explanation / Answer

Specific activity of an enzyme is defined as the enzyme activity per unit total enzyme present i.e.

Specific activity = Enzyme activity/Total mass of protein or enzyme

Thus, the answers can be found as below:

Part 32:

a) Specific activity of enzyme after step 1 :

Enzyme activity/Total protein or 1*1010/5*105 or 2*104 units/mg protein

b) Total yield:

After step 1: Total yield = Enzyme units activity/specific activity or 8*109 / 8*104   or 105 mg.

After step 2: Total yield = Enzyme units activity/specific activity or 5*106 / 5*105   or 10mg.

c) Overall percentages:

Percentage yield: (Total yield*100)/Yield after final purification

i.e. 20*100/100000 or 0.02%

Specific activity after final purification = 4*104 / 20 or 2*103 units/mg protein.

Thus, percentage change in specific activity = (2*103)*100/2*104 or 10%

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