Chapter 8: Multiple Choice Questions

Question 1

For an application where you require a sample of your target protein at high purity,
what would be a good purification strategy? Assume that your starting point is E. coli
cells in which the target protein fused to an affinity tag has been over-expressed.

a) Affinity chromatography (AC) followed by size exclusion chromatography (SEC)

b) AC only
c) AC followed by ion-exchange (IEC) followed by SEC
d) AC followed by IEX, followed by hydrophobic interaction (HIC) and then SEC

Feedback:
For applications requiring the purest protein, you will usually start with a protein over-
expressed in e.g. E. coli with an affinity tag, so affinity chromatography will be the first step.
Although this is efficient, adding an extra step or steps (for intermediate purification or
polishing) will be useful to remove impurities or mis-folded protein. Too many steps though
can lead to poor yield, as some protein is lost with each purification step.

Question 2

You know that the protein you want to purify from a natural source forms a multimer
with multiple sub-units giving a molecular weight in solution much bigger than
visualised denatured on SDS-PAGE. There is only a small amount of the target protein
in the total protein sample. Which of the following is an appropriate purification
strategy?

a) SEC
b) IEX followed by SEC
c) IEX
d) AC followed by SEC

Feedback:
 The molecular weight of the protein in solution, suggests that SEC would be appropriate.
However, the filtration properties of SEC matrices compared to the capture properties of
IEX or AC matrices means that only small sample volumes can be loaded on SEC columns;
typically ~1% of the column volume. As your target protein is already quite dilute, an
unreasonably large column would be necessary for purification of a useful amount of
protein. SEC also dilutes samples further during chromatography. A "capture" step such as
IEX, AC or HIC would be a good first step to concentrate the protein, while also removing
impurities. As the protein is to be purified from a natural source, the affinity chromatography
matrices used for recombinant proteins will not be suitable (but there may be a more
unusual affinity matrix specific for your protein). IEX is a good capture step. Adding a SEC
step gives additional purification using a different property of the protein.

Question 3

You find that your protein sample loses activity during storage. What can you do about
this?

a) Add an additional purification step

b) Use a protease inhibitor during purification steps
c) Perform each step as quickly as possible, in a cold-room
d) All of the above

A protein sample might lose activity for many reasons, such as proteolysis or
aggregation. Adding an additional purification step will likely remove protease
impurities. Including additives such as protease inhibitors or reducing agents can help
protect your protein against proteolysis or oxidation. Others steps which can improve
purity and stability are also possible.

Question 4

Question 4
 Which of these techniques is often considered a suitable "polishing" step in a protein
purification strategy?

a) Affinity chromatography (AC)

b) Ion-exchange chromatography (IEC)
c) Hydrophobic interaction chromatography (HIC)
d) Size-exclusion chromatography (SEC)
Purification strategies comprise of capture steps, intermediate purification and polishing steps
(SEC). SEC is often used as a final step to remove any last, minor impurities and mis-folded
protein and hence a suitable polishing step.
Question 5

Which of these chromatography types are suitable as a "capture" step in the

purification of non-tagged proteins?

a) SEC
b) Dialysis
c) IEC and HIC
d) Ammonium sulphate precipitation

Feedback:
A capture step is used as the first step of a purification strategy where the goal is to remove
quickly as many impurities as possible using a relatively cheap, high-capacity matrix. IEX
and HIC are good capture steps. Affinity chromatography is also often used as a capture
step for affinity-tagged proteins. SEC is too slow (exposing your protein to proteases) and
the column too expensive to be used as an early purification step where the column might
become blocked by cellular debris in the extract. Dialysis is often used between steps to
change the solution conditions (e.g. salt concentration) between purification steps.
Ammonium sulphate precipitation is often used as a first non-chromatographic purification
step to clean-up an extract before chromatography.
Question 6

Nickel-NTA (Ni-NTA) chromatography is a popular affinity chromatography method for

the purification of histidine-tagged proteins. However, SDS-PAGE of the eluted protein
can show bands in addition to your target protein. How might you improve this
purification step?

a) Add a low concentration of imidazole to the binding buffer

b) Gradient elution with increasing imidazole concentration on a chromatography
machine
c) Include protease inhibitors during purification
d) All of the above

Feedback:
Some proteins from the expression host can also bind to a Ni-NTA matrix. Adding low
concentrations of imidazole to the sample when passing the extract through the column can
reduce this non-specific binding, without affecting the binding of the target protein to the
column. Gradient elution can wash out weakly-bound impurities before the target protein is
eluted at higher imidazole concentrations. However, if you only see bands smaller than the
expected size of your target protein, these may be proteolysis products of your protein.
Adding a protease inhibitor cocktail can prevent or reduce this. An additional purification
step may be necessary to improve purity.

Question 7

What is the starting point for selection of a suitable IEX matrix for purification of a
recombinant protein?

a) Prediction of isoelectric point (pI) from the amino acid sequence

b) Test protein binding to an IEX matrix at a range of pHs and salt concentrations
c) Test protein binding to a selection of anion and cation exchange matrices
d) Pass your sample through a preparative column and elute with a salt gradient
 Feedback:
If there is a crystal structure or NMR structure of the protein available you will know how the
charged amino acids are distributed around the protein, otherwise not. In the latter case you
may need to do some empirical tests of binding using different matrices under different
conditions. However, as you have a recombinant protein expression vector, the amino acid
sequence will be known hence an isoelectric point using Web-based tools can be predicted.
This can give a useful indication of which matrix to use. You might go straight to a
preparative column if you have no information about the protein's activity - in this case,
remember to test flow-through and elution fractions for activity.

Question 8

Your purification strategy of combinations of chromatography steps gives a protein

preparation with a single band on SDS-PAGE. Which of the following would be best for
determining the protein concentration (as mg/ml or molarity)?

a) Measure a UV absorbance scan and use the absorbance at 280nm with the molar
extinction coefficient (predicted from the amino acid sequence)
b) Determine amino acid composition after hydrolysis to amino acids
c) Colorimetric assay using Bradford or BCA assays
d) "Guesstimate" the concentration from the intensity of bands on SDS-PAGE gels

Feedback:
The concentration of purified proteins is required for most applications - at least so you can
compare results between experiments with different batches of protein. As well as
measuring the absorbance of the sample at 280nm, a UV absorbance scan can provide
additional information such as any indication of protein aggregation or the presence of some
impurities. Protein concentration can be calculated using the A280 absorbance reading and
the molar extinction coefficient using the Beer-Lambert law. Dialysis may be necessary to
remove impurities such as imidazole which will add to the absorbance. Colorimetric assays
are commonly used and easy to use. However, check for any compounds which might
interfere with the assay. In addition, choice of protein to use for the absorbance calibration
curve can affect the accuracy of the concentration determination of the target protein.
Sometimes you will need a very
Question 9

What properties of a protein does hydrophobic interaction chromatography exploit for

purification?

a) Charged amino acids

b) Hydrophobic amino acids on the protein surface
c) Molecular weight
d) Enzyme activity

Feedback:
Hydrophobic interaction chromatography (HIC) uses hydrophobic amino acids on the
surface of the protein to interact with a matrix carrying other hydrophobic groups, such as
butyl or phenyl. IEX uses interactions between opposite charges on protein and column
matrix. SEC separates molecules based on molecular weight and shape. Substrate
specificity may be exploited by affinity chromatography. Use different types of matrix
exploiting different properties - impurities are unlikely to share the same properties as your
target prote

Question 10

To elute target proteins from an affinity chromatography matrix, which of the following
conditions would be the most appropriate?

a) Low salt concentrations

b) High salt concentrations
c) Adding a soluble ligand which competes with the affinity tagged protein for
binding to the column
d) Just keep washing buffer through the column, isocratic elution

Feedback:
 Affinity chromatography exploits the specificity of a protein for binding only certain other
molecules. Immobilising one molecule on a chromatography matrix allows you to capture a
binding partner. A soluble ligand passed through the column can bind the target protein,
displacing it from the column so it can be washed out. However, some interactions are very
strong and require more aggressive conditions; very low pH is often used to disrupt
antibody-antigen interactions and for Protein A purification of antibodies. High salt
concentrations are used to elute proteins from IEX columns, while low salt concentrations
are used to elute proteins from HIC columns. Isocratic elution is used for SEC - washing an
affinity column without changing binding conditions will not elute your protein.

Question 11

Which of the following methods could be used to check the molecular weight of your
purified protein?

a) SDS-PAGE only
b) Mass spectrometry only
c) Analytical SEC only
d) All of the above.

Feedback:
SDS-PAGE is used to check the progress of protein purification. Electrophoresis of a set of
molecular weight standards alongside the target protein allows you to check if its molecular
weight matches the weight expected. If it does not match, the bands may be impurities or
proteolysis products. Remember that proteins with high proportions of acidic or basic amino
acids can appear lower or higher molecular weight respectively than would be expected.
Mass spectrometry can provide a much more accurate molecular weight than SDS-PAGE,
indicating any proteolysis or modification. In contrast to the denatured molecular weight
from SDS-PAGE, analytical SEC provides the molecular weight of the native protein,
revealing any protein multimerization.