Bke2 Biochemistry Exercises

Group exercise: Biochemical methods
In this exercise, we will take a walk through an imaginary protein preparation and analysis (of proteins X and Y), looking at which steps might typically be used, how they work, and how they are linked together.

One thing to note before we get started, is that biochemists often refer to a protein's size in terms of its molecular weight, in kDa (a kilodalton, kDa, is 1000 times the molecular mass of hydrogen). Each amino acid residue counts for about 110 daltons, that is, about 0.11 kDa.

Let's pretend that we had grown some cells, and from all of the proteins in the cell we want to purify two proteins, let's call them X and Y. We have broken the cells open with sonication (homogenisation with ultrasound) in a buffer at pH 7, and removed the cell debris (membranes, cells walls, etc.) by centrifugation. We will use (NH₄)₂SO₄ precipitation as our first purification step here. It is a crude method of protein fractionation, usually applied in the early stages of purification. We add (NH₄)₂SO₄ to our protein solution to 40% saturation (about 1.6 M (NH₄)₂SO₄), and centrifuge out anything that precipitates then. To the remaining solution, we'll add (NH₄)₂SO₄ to 70% saturation, and spin down things that precipitate then. The two "pellets" (the stuff that precipitated out), are redissolved in a small volume of buffer. We'll keep each of these three samples separate.

a) Describe the principle of (NH₄)₂SO₄ fractionation. Would you expect all proteins to precipitate at the same concentration of (NH₄)₂SO₄? Why or why not?

b) Suggest two methods for removing (NH₄)₂SO₄ in preparation for the next purification step. Do you actually need to remove the (NH₄)₂SO₄ before the next purification step given here (see Step 3)?

Next, we'll use SDS-polyacrylamide gel electrophoresis (SDS-PAGE) to analyze our three samples from the (NH₄)₂SO₄ fractionation. We find that both X and Y came out in the same ammonium sulfate fraction (between 40%-70% saturation). So, they will need further purification, if we want them separated from each other, and from other proteins.

We can also use this method to measure the molecular weight of the denatured proteins, if we know how a set of standard proteins behaves (we run samples of them at the same time, on the same gel).

The following proteins were subjected to SDS-PAGE:

Protein	M_r(kDa)	migration (mm)
serum albumin	67	11
ovalbumin	45	23
carbonic anhydrase	32	34
trypsin inhibitor	21.5	46
lysozyme	14.4	59
Protein X	?	28
Protein Y	?	54

Plot the log of the molecular weight (in kDa) versus the distance the protein migrated in the gel. That plot should give a straight (well, almost) line for the known proteins. Use this plot to estimate the molecular weights of Proteins X and Y.

Gel filtration chromatography can be used both for purification, and to estimate the native (non-denatured) molecular weight of proteins. So, we will use it to determine the molecular weights of X and Y, at the same time as we purify them further. To do the size estimate, we need to know the relationship between protein size and behavior on a particular gel filtration column. We, therefore, "calibrate" our column, by passing a series of proteins with known molecular weights through it, measuring the elution volume for each protein (V_e, the volume of solution it takes to elute - wash off - that protein from the column). A value, K, can then be calculated for each protein, using the formula:

K = (V_e - V₀)/(V_T - V₀)

where

V_e = elution volume
V_T = total column volume
V₀ = void volume

(You obviously need to have V₀ and V_T as well as V_e, to use the formula.) A plot of log(M_r) versus K should yield a straight line. The molecular weight of any other protein can then be determined from its elution volume, together with this plot. So, let's get V_T and V₀.

a) The void volume (V₀) is the volume of the fluid outside of the resin beads. How might one determine the void volume of a gel filtration column?

b) Our gel filtration column has a diameter of 7.5 mm and a length of 300 mm. What is the total volume (V_T) of the column?

c) Now this column can be used to estimate the molecular weight of our two proteins, X and Y. Given that V₀ of the column = 5.2 ml, the V_T from part b, and the elution volumes for a series of proteins as follows:

Protein	M_r (kDa)	V_e (ml)
thyroglobulin	669	7.7
catalase	232	9.4
alcohol dehydrogenase	150	9.8
serum albumin	67	10.0
ovalbumin	43	10.7
RNase	13.7	12.0
Protein X	?	9.9
Protein Y	?	10.3

calculate the K's and make a plot as described above. What are the measured molecular weights of Proteins X and Y by this method?

d) In the SDS gel, which protein ran fastest? Is that the same as their order of elution on the sizing column here? Why or why not?

e) Compare the values you obtained for the molecular weights of X and Y with the results you obtained from the SDS gel. Suggest a possible reason why these results may not agree. Is there a better way to determine the molecular weight of a protein?

To choose further purification methods, it can be very helpful to know something about the charge characteristics of your proteins, and how different pH's may affect them. In the example given here, a special gel is used to determine the charge of a protein at a variety of pH values. The gel has a pH gradient from 3 to 9 along the x-axis:

A line of protein solution is applied to the gel (along the dashed line in the diagram). An electric potential is applied to the gel, and the proteins migrate towards the anode (+) or cathode (-), depending on their charge.

a) What are the approximate pI values of Proteins X and Y?

b) The charge of a protein affects its solubility. We used solubility as a means of crude purification in Step 1. Which pH values might be used for optimizing separation of X and Y by (NH₄)₂SO₄ precipitation?
Ion exchange is one of the most useful of the column chromatography methods, and we'll do that next.

a) What pH value would be suitable for chromatography of Y on DEAE-agarose (anion-exchange; see p. 66 of Horton or p. 50, Stryer)?

b) What pH value might you choose for chromatography of Y on CM-Sepharose (cation-exchange)?

c) What kind of ion exchange and what pH will you use to separate proteins X and Y? How will you elute the proteins from the column?
These days, we often try to get smarter when we need to prepare a new protein. One useful way is to "fuse" our protein sequence to another one that we can use as a handle for purification in affinity chromatography, often called a purification "tag". A very common tag is the enzyme glutathione transferase (GST). In vivo, GST catalyses the attachment of glutathione (a small peptide found in most cells) to a variety of other molecules (often toxic compounds), as the first step of getting rid of them. GST has a high affinity (= strong binding) for glutathione, which we can make use of here.

So, let's pretend that we have constructed a gene for such a fusion protein by joining the gene sequences for GST and protein X. We also put a short gene sequence in between that codes for a short linker peptide that contains a cleavage site for a protease. We put the gene in a bacterium (eg. E. coli) that reads the gene and makes a single polypeptide that contains both the GST enzyme and our protein X and has a linker peptide in between the proteins that can be recognised and cleaved by a protease. We have got our fusion protein:

Let's suppose that you found that protein Y precipitated irreversibly when you used the (NH₄)₂SO₄ step. Suggest another purification protocol for our proteins X and Y. This is limited only by your own creativity!

Reading material: Horton, Chapter 3 (also try Stryer, Chapter 3, if you want more reading)

Lecture:
Biochemical methods

Links:
Suggested answers

Exercise index

[1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14]

Exercise by Markku Saarinen & Sherry Mowbray
Contents updated 2000.08.22 by mowbray@xray.bmc.uu.se
Page updated 2002.09.04 by jerry@xray.bmc.uu.se
Copyright © 1998-2000. Department of Molecular Biology SLU. All rights reserved.