Question

1. During the course of a protein purification, we usually make a purification table to help keep track of the success of the process as we proceed from crude homogenate to the final product. During enzyme purification, the important quantities to keep track of are listed on the table given below. For example, the following enzyme (enzyme X) is positively charged. For its purification, a recombinant protein was created by adding a c-myc tag to the gene. It was purified following these steps: Complete the following purification table, including missing units. (1 point) Procedure After these steps Total Proteins (mg) Enzyme X % (mg) Yield 1. Crude Extract 1000 200 100 2. Size exclusion 900 50 25 3. Ion- exchange 500 40 20 Chromatography 4. Affinity 37 32 16 Chromatography 5. Salt precipitation 28 5 2.5 (a) From all the purification steps listed above, which one (s) is/are necessary? (1 point) (b) Which, if any, of the steps would you eliminate, and why? (1 point) (c) Specify which type of resins would be used for the purification of the enzyme: For steps-3 (ion exchange chromatography) and 4 (affinity chromatography). (1 point)

          1. During the course of a protein purification, we usually make a purification table to help keep
track of the success of the process as we proceed from crude homogenate to the final product.
During enzyme purification, the important quantities to keep track of are listed on the table given
below. For example, the following enzyme (enzyme X) is positively charged. For its purification,
a recombinant protein was created by adding a c-myc tag to the gene. It was purified following
these steps:
Complete the following purification table, including missing units. (1 point)
Procedure
After these steps
Total
Proteins
(mg)
Enzyme X
%
(mg)
Yield
1. Crude Extract
1000
200
100
2. Size exclusion
900
50
25
3. Ion- exchange
500
40
20
Chromatography
4. Affinity
37
32
16
Chromatography
5. Salt precipitation
28
5
2.5
(a) From all the purification steps listed above, which one (s) is/are necessary? (1 point)
(b) Which, if any, of the steps would you eliminate, and why? (1 point)
(c) Specify which type of resins would be used for the purification of the enzyme: For steps-3 (ion
exchange chromatography) and 4 (affinity chromatography). (1 point)

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1. During the course of a protein purification, we usually make a purification table to help keep
track of the success of the process as we proceed from crude homogenate to the final product.
During enzyme purification, the important quantities to keep track of are listed on the table given
below. For example, the following enzyme (enzyme X) is positively charged. For its purification,
a recombinant protein was created by adding a c-myc tag to the gene. It was purified following
these steps:
Complete the following purification table, including missing units. (1 point)
Procedure
After these steps
Total
Proteins
(mg)
Enzyme X
%
(mg)
Yield
1. Crude Extract
1000
200
100
2. Size exclusion
900
50
25
3. Ion- exchange
500
40
20
Chromatography
4. Affinity
37
32
16
Chromatography
5. Salt precipitation
28
5
2.5
(a) From all the purification steps listed above, which one (s) is/are necessary? (1 point)
(b) Which, if any, of the steps would you eliminate, and why? (1 point)
(c) Specify which type of resins would be used for the purification of the enzyme: For steps-3 (ion
exchange chromatography) and 4 (affinity chromatography). (1 point)

Added by Howard D.

Biology for AP Courses

Julianne Zedalis, John Eggebrecht

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Solved by Expert Dennis Howard

07/27/2022

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During the course of a protein purification, we usually make a purification table to help keep track of the success of the process as we proceed from crude enzyme homogenate to the final product. The important quantities to keep track of are listed on the table given below. For example, the following enzyme (enzyme X) is positively charged. For its purification, recombinant protein was created by adding a myc tag to the gene. It was purified following these steps: Complete the following purification table, including missing units: (1 point) Procedure After these steps | Total Proteins (mg) | Enzyme X (mg) | % Yield 1. Crude Extract | 1000 | 200 | 100 2. Size exclusion | 900 | 50 | 25 3. Ion-exchange Chromatography | 500 | 40 | 20 4. Affinity Chromatography | 37 | 32 | 16 5. Salt precipitation | 28 | 5 | 2.5 (a) From all the purification steps listed above, which one(s) is/are necessary? (1 point) (b) Which, if any, of the steps would you eliminate, and why? (1 point) (c) Specify which type of resins would be used for the purification of the enzyme: For steps 3 (ion-exchange chromatography) and 4 (affinity chromatography). (1 point)

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Transcript

00:01 This question has three parts.

00:02 The first part of the question, part a, asks us to determine which of the purification steps are necessary.

00:11 So the way that you would approach this is you'd want to determine the change in your total protein mass versus the change in your enzyme, either by looking at the enzyme mass or the percent yield.

00:30 And from there determining whether the change was sufficiently significant to actually go through that processing step.

00:44 So for example, going from crude extract to size exclusion, we only lose 100 milligrams of our total protein.

00:52 So that's a 10%.

00:54 It's a 10%.

00:55 So perhaps what we can do is we can just draw out this table without writing it, but we'll just do the percentage.

01:01 For the percent change.

01:05 So we can do essentially 900 over 1 ,000.

01:15 And then the next step would be, so this would be like 1.

01:18 2, we would have 500 over 900.

01:22 3, we'd have 37 over 500.

01:28 And then 4, we'd have 28 over 37.

01:32 And we're going to compare that to our 25%, our 20%, our 20%, 16%, 2 .5%.

01:46 So the percent yield is essentially telling us how much we've lost of our target versus this left column is telling us how much we've lost of our total protein.

02:08 So we'll see in just a moment what this all means.

02:11 So here we have 90 % still remaining.

02:14 Then if we do the 500 over 900 is about.

02:18 55 or 56%.

02:21 Then we do 37 over 500 is going to be 7 .4%.

02:29 And then finally, 28 over 37 gives us 76%.

02:42 So between steps 1 and 2, we are left with 90 % of our total protein.

02:52 So we only lose 10%.

02:54 Whereas we lose 25 % of our initial target enzyme then going on to number two so let's just erase this part a here because we'll answer part a and b at the same time so going on to this number two between steps two and three we lose 44 percent of our protein and we lose 20 percent of our target protein our target and then we lose quite a lot right we do 7 .4 that's 92.

03:29 Of our total protein that we've lost, and we've only lost 16 % of our total enzyme.

03:35 And then finally we lose 76%, we are left with 76%, so we lose only 24%...

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