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LAB 4A - Computer Simulation of Protein Purification
Objective
In lab 4 you will be introduced to classical protein purification. Protein purification
reduces the amount of, or eliminates, contaminating proteins from the protein of
interest. There are two different approaches to this. In the classical method, protein is
naturally expressed and purified from the original organism. For proteins expressed
from the original organism the purification strategy depends upon the physio-chemical
properties of the protein such as size, charge, shape, polarity, or any innate, unique
binding properties. A more recent method, based on recombinant DNA technology,
purifies protein that has been expressed from a gene cloned into a specialized plasmid
vector. Choice of method depends on a number of factors including how well
characterized the organism is and the nature of the protein of interest. Cloning is an
efficient method of protein expression and purification. However, it isn’t always an
option. If, for example, the gene is not easily cloned, or if the expressed protein isn’t
functional when cloned, the classical method must be used.
In Lab 4A you will perform a computer simulated classical protein purification. This
will familiarize you with the process before you perform the -galactosidase
purification experiment in Lab 4B. The computer program simulates the experience of
working through a protein purification problem using a variety of separation and
analytical techniques. It provides insight into the strategies commonly employed in
protein purification without actually performing the experiments, thus saving time and
materials. Practical experience is of course still important, but the strategies involved
will be better learned and more meaningful with an initial grounding in the
fundamental principles.
Procedure
Week 3, Day 2
Meet in the Clearihue A012 computer lab for this lab session.
In this simulated protein purification exercise, each student will be assigned 1 protein to
purify. Your task is to purify your assigned protein to homogeneity in the most effective
and efficient manner possible. You will be able to access the program on these
computers by following the instructions given in Part A.
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Note: if you must complete this exercise outside of this lab session, you can access the
program from any computer with an internet connection. However, you may need to
download Java software onto your computer. To do this:
1. Go to http://java.com/en/download/windows_ie.jsp?locale=en&host=java.com and download
the Java software.
2. Proceed with Part A.
A. Purifying the Proteins
1. Go to http://www.bio-link.org/clearinghouse/protein.htm
2. In the Introduction section “About the Software…” click on the following link
http://www.booth1.demon.co.uk/archive
3. Choose the Java version.
4. A page will open entitled “The Strategy of Protein Purification”. About 2/3 down the
page is the sentence “Make sure you have the Java Plug-in installed and then click
here to start the applet in a new window”. Click as indicated and the Protein
Purification Program will open.
5. Before you begin purifying your protein, read the information under “Strategy” in
the “Help” menu. This will help you get started and give you an overview of how to
choose purification methods.
6. Choose “Start from the beginning” from the “Start” menu.
7. Choose “Default_Mixture” and click “OK”.
8. Enter the protein number that you have been assigned and click “OK”.
9. Record the initial information given to you about this protein and click “OK”.
10. Look at the Coomassie and immunoblotted gels to determine the approximate
molecular weight and pI of your protein. To do this, go to the “Electrophoresis”
menu and select “2-Dimensional PAGE”. Then, again under the “Electrophoresis”
menu, select “Immunoblot”. When you have recorded the pI and molecular weight
of your protein, choose “Hide immunoblot” from the “Electrophoresis” menu.
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11. Choose a separation method under the “Separation” menu. A brief description of
each method is given below. A more comprehensive description can be found by
clicking on “Info” after a separation method has been chosen. Be sure to record
which separation methods you choose and the conditions under which you chose to
run the experiment so that another person could repeat the purification exactly as
you performed it and get the same results.
a) Ammonium Sulphate Fractionation
 “salting out” of protein fractions is based on solubility.
 often used as an initial purification technique.
 choose the final salt concentration to precipitate the protein of interest (or
you may want to precipitate the contaminant proteins while the protein of
interest stays in solution).
b) Heat Denaturation
 separates proteins on the basis of heat stability.
 only useful for proteins stable at high temperatures (ie. 50oC or above)
c)
Gel Filtration
 separates proteins on the basis of size ( ie. larger proteins are eluted before
smaller proteins).
 You must select an appropriate gel filtration media. Initially, aim to have the
molecular weight of your protein in the middle of the size range.
 The following table gives the gel filtration matrixes and their size ranges:
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Characteristics of gel filtration media used in this simulation
Matrix name
Bead type
Sephadex G-50¹
Sephadex G-100¹
Sephacryl S-200 HR¹
Ultrogel AcA 54²
dextran
dextran
dextran
polyacrylamide/
agarose
polyacrylamide/
agarose
polyacrylamide/
agarose
polyacrylamide
polyacrylamide
polyacrylamide
Ultrogel AcA 44²
Ultrogel AcA 34²
Bio-Gel P-60³
Bio Gel P-150³
Bio-Gel P-300³
Approximate fractionation
range for peptides and globular
proteins (molecular weight)
1500 - 30000
4000 - 150000
5000 - 250000
6000 - 70000
12000 - 130000
20000 - 400000
3000 - 60000
15000 - 150000
60000 - 400000
¹Sephadex is a registered trademark of Pharmacia PL.
²Ultrogel is a registered trademark of Pharmacia-LKB.
³Bio Gel is a registered trademark of Bio-Rad Laboratories, Inc.
d) Ion Exchange Chromatography
 separates on the basis of charge
 there are four matrices to choose from in this program
i) DEAE-cellulose: anion exchange column
ii) CM-cellulose: cation exchange column
iii) Q-Sepharose Fast Flow: strong anion exchange gel; retains its positive
charge over a wide pH range
iv) S-Sepharose Fast Flow - strong cation exchanger
 choose a column matrix and equilibrate using a buffer with a suitable pH
(start with a neutral pH and work from there).
 choose between a gradient of increasing salt concentration or a pH gradient
to elute proteins from the column.
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e) Hydrophobic Interaction Chromatography
 separates proteins on the basis of interactions between hydrophobic residues
or “patches” on the protein surface and the hydrophobic groups on the HIC
matrix. The initial high salt conditions enhance the interactions by removing
water molecules from the protein surface. The descending salt gradient
allows the proteins to rehydrate and leads to their selective elution with
retention of biological activity.
 You are given a choice of 2 hydrophobic gels:
i)
Phenyl Sepharose CL-4B – a less hydrophobic matrix which separates
strongly hydrophobic proteins.
ii) Octyl Sepharose CL-4B – a more strongly hydrophobic gel, which
separates weakly hydrophobic proteins.
 You must also establish a descending salt gradient.
f) Preparative Isoelectric Focusing
 Separates proteins on the basis of charge.
 You must choose the pH range for the gradient. Aim to have the pI of your
protein in the middle of this range.
g) Affinity Chromatography
 separates proteins based on their affinity for a specific ligand that is
immobilized on a solid support.
 You have the choice of using one of the following ligands:
i) 3 types of monoclonal Ab. (low, medium or high affinity)
ii) Polyclonal IgG preparation
iii) For some proteins, a competitive inhibitor is available.
 You must also choose the elution method.
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12. After each separation step, analyze the success of the chosen technique:
i) Determine the fractions with enzyme activity (not necessary for ammonium
sulphate fractionation or heat denaturation). In the “Fractions” menu click on
“assay enzyme activity”. The region highlighted in red indicates the fractions
with enzyme activity.
ii) Pool the fractions that you judge to have significant activity by choosing “Pool
fractions” from the “Fractions” menu, and select the fractions you want pooled.
iii) Record the “situation” given (total protein, total enzyme, enrichment, yield, and
cost), and click “OK”.
iv) Determine the purity of the pooled fractions by 1D or 2D electrophoresis,
whichever you judge to be most appropriate.
13. Choose the next separation technique in your purification scheme based on the
information from the gels.
14. Continue until you have purified your enzyme to homogeneity. This will be
confirmed by the presence of only 1 band (in certain cases, 2 bands) on the 1D and
2D gels. A summary of your purification steps including the total protein, enzyme
activity, enrichment, % yield and man hours can be found in “Progress Report”
under the “Help” menu.
15. Repeat the purification process, trying different separation techniques or different
parameters, to find the methods and experimental parameters that give better
purification results.
B. Documenting and Fine-Tuning the Purification Procedure
1. Create a record of the purification procedure that gave you the best results. To do
this repeat the purification steps without running any gels (gels decrease yield and
increase man hours).
2. After the final step, choose “Progress Report” from the “Help” menu. A summary of
the purification at each step will be displayed including the total protein, enzyme
activity, enrichment, % yield, and cost (man hours). Print a copy of this table by
pressing the Print Screen (PrtScn) button on your keypad and then paste it into a
Word document. Include this with your summary.
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3. Print a copy of the Coomassie stained 1D and 2D gels to prove the purity of your
protein. To do this, press the “PrtScn” button on your keypad and then paste it into
a Word document.
4. Your purification strategy will be assessed by comparing it with “optimal” values.
These will be posted on Blackboard for you to see.
Summary - Lab 4A
For your assigned enzyme, include the following information:
1.
2.
3.
4.
5.
6.
7.
Enzyme number
Stability information
Molecular weight
pI
Any other pertinent information (e.g. ammonium sulfate concentration,
fractions pooled, pH of buffers, elution concentrations, etc.) to allow the marker
to repeat the purification exactly as you performed it and arrive at the same
enrichment value and % yield.
A print out of the final “Progress Report”.
A print out of the 1D and 2D gels showing the purified protein. (In most cases
the gel will show only one band, but in some it may show 2).