Chemistry 394 – Spring 2015
Experiment 1 – Purification of Ulp1
Background
Protein purification is the among the most fundamental activities associated with biochemistry.
Without protein there is often nothing else to study. In this experiment, you will use immobilized
metal ion affinity chromatography (IMAC) to purify Ulp1, a sequence-selective endoprotease, from
a recombinant strain of E. coli.
IMAC is currently the most commonly used form of chromatography in purification. Genes for a
protein of interest are typically cloned into an expression vector that attaches a short stretch of DNA
encoding 6-8 histidine residues at either the N- or C- terminus of the protein (a so-called fusion His
tag). The histidine residues ligate various metal ions with varying affinity, and provide a specific
chemical interaction that can be used to separate them from other proteins in the cell on the IMAC
column. In this experiment, we will use immobilized cobalt(II) or nickel(II). Cobalt tends to form
weaker interactions with histidine residues leading to fewer contaminants that bind adventitiously,
but the yield of the desired protein can be lower than when nickel is used. Since nickel interacts
strongly with histidine residues the His tag sticks pretty well to it, but so do some other native E. coli
proteins.
The protein of interest (POI) in this experiment is the ubiquitin-like-specific protease 1 (Ulp1).
Ulp1 is a reagent itself in protein expression and purification. There are two common problems in
protein expression and purification of fusion proteins. One is that the fusion protein is ideally
expressed in a soluble form, and second is that the POI be separable from any fusion partners in
use. The SMT3 protein from yeast is a small (11.5 kDa) single domain monomeric protein that is
both stable and soluble. Ignoring its biological purpose, from a biotechnology purpose it can be
placed in the same reading frame as a POI to assist in solubilizing the latter. Place a hexahistidine tag
at the N-terminus and one has a stable fusion protein that will selectively bind to an IMAC column.
To cleave the His6SMT3 fragment from the fusion protein one uses the highly select Ulp1, which
cleaves after the last glycyl residue of the SMT3 domain to yield two fragments – the His6SMT3
protein and the POI.
Ulp1 is a cysteine protease, using an active site cysteine residue as the nucleophile for initial attack
on the SMT3 fusion protein (***). The protein we are purifying has been modified via molecular
biology to only incorporate the C-terminal protease fold (residues 432-621) and to incorporate a
His-tag at its N-terminus, just like the SMT3 fusion protein. That His tag makes Ulp1 easy to purify
and also easy to remove from a reaction between Ulp1 and a fusion protein.
For your convenience, I have already obtained a recombinant strain of E. coli expressing His tagged
Ulp1. Your job is to perform the following operations over the coming weeks: (1) separation, (2)
determination of purity, (3) quantitation and (4) determination of activity. A brief schedule of
operations follows:
Week 1: Preparation of materials (buffers, gels)
Week 2: Cell lysis, column chromatography, dialysis
Week 3: Gel electrophoresis, protein concentration assay, protein activity assay(s)
Experimental
Week 1:
Buffer preparation
You and your partners are tasked with prepared the two buffers that are required for purification:
500 mL Purification buffer: 50 mM Tris, 350 mM NaCl, 10 mM imidazole, 0.2% triton X-100,
pH=8. (you will need to add β-mercaptoethanol to 1 mM next week)
500 mL Storage buffer: 25 mM Tris, 250 mM NaCl, 50% glycerol, 1% triton, pH=8. (you will need
to add DTT next week)
Also…
Pouring a gel
Pour one Laemmli discontinuous gel. Buffers and reagents are available in lab and a helpful video is
posted on the web page. Please note that acrylamide is a neurotoxin and suspected carcinogen. Wear gloves while
preparing the gel.
Week 2:
Purification
You will be provided a frozen pellet of E. coli cells (hopefully) containing Ulp protease:
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Buffers
Reducing agents need to be added to both buffers. β-Mercaptoethanol should be added to
the purification buffer (1 mM) and dithiothreitol, or DTT, to the storage buffer (0.5 mM).
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Resuspension
Add enough lysis buffer (purification buffer + 20 % sucrose) to your cell pellet to bring the
volume to 25 mL. Add a small amount (~1 mg) of lysozyme to the buffer. Resuspend the
pellet by pipetting up and down with a 10 mL pipet. Return the pellet to ice to cool for a
few minutes.
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Lysis
Lysis will be performed by sonication. We have two sonicators – depending on how things
go you will either (a) transfer the cell suspension to a 30 mL beaker on ice or (b) transfer the
cell suspension to two 15 mL falcon tubes. I will offer explicit instructions on how to run
the sonicators.
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Clarification of lysate
Remove 25 µL of lysed cells to a microcentrifuge tube and add 25 µL of 2x SDS loading
buffer. This sample will be used for SDS PAGE. Transfer the remaining cell lysate to a 40
mL centrifuge tube and cap it. Take another centrifuge tube with cap, and balance it with
DI water. Centrifugation will be performed at 15,000 rpm in an SS34 rotor for 10 minutes
at 4˚C. Take note of the gravitational force – this will be what you report in your write-up.
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Preparation of chromatography set-up
While the centrifuge is running, pump purification buffer through your column to pre-equilibrate
it to the buffer. Also prepare a 50 mL sample of elution buffer (purification buffer + 250
mM imidazole).
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Application of clarified lysate to column
Remove tubes from centrifuge and decant supernatant into a clean tube. Reserve 25 µL of the
clarified lysate for PAGE and 200 µL for the protein and activity assays next week. Insert the inlet
tube from the column into your lysate and load it on the column using the pump. Start
collecting 5 minute fractions on your carousel.
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Column Wash and Elution
When all the lysate has been added, elute unbound protein with 15 mL of purification
buffer. Maintain 5 minute fractions for this part of the chromatography experiment.
Following the wash, set the fraction size to 3 minutes and elute the column with a 30 mL
elution buffer (purification buffer + 250 mM imidazole).
Occasionally, transfer tubes from the carousel to an ice bucket, being sure to label each tube
with its fraction number.
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Testing fractions for protein
Take a piece of filter paper and number it from 1 to your highest fraction number. Spot 2
µL of each fraction on the corresponding location. Dry the paper with a heat gun. Briefly
stain with Coomassie blue solution and rinse with water. Note that the fractions of interest
will be those containing protein following initiation of the gradient.
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Dialysis
If the dot test looks appropriate, you will dialyze fractions you suspect contain Ulp1
overnight against 500 mL of storage buffer (with the added DTT) at 4˚C with stirring. I will
provide detailed instructions for this setting up dialysis in lab.
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Cleaning the column
When the gradient is finished, elute the column with 30 mL of purification buffer (Tuesday
lab) or 30 mL diH2O then 30 mL 20% ethanol (Wednesday lab). Stop the pump and cap
both ends of the column.
Week 3
Activity Assay
In any enzymatic purification, it is expected that one will assay the activity of the purified protein.
This will be done by monitoring proteolytic cleavage via gel electrophoresis and fluorescence
anisotropy in the Beacon 2000 fluorescence polarization instrument.
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Activity measured by gel electrophoresis. I will provide 50 µL of a SMT3 fusion protein to
you (MW = 40 kDa). Incubate 25 µL of the fusion protein with 5 µL of your Ulp1 for 30
minutes at room temperature. You will load both the reacted and unreacted fusion protein
on your gel (below).
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Continuous activity measurement
The assay will be performed in a buffer I provide (25 mM HEPES, pH 75, 200 mM NaCl
and 1 mM β-mercaptoethanol). Add 1 mL to the provided test tube and place it in the
Beacon 2000 instrument to blank. Following the blank, add 2 µL of the fluorescently labeled
substrate (stock solution is 100 µM in the substrate), vortex gently and measure the anisotropy
at time zero. Then remove the sample and add 25 µL of your Ulp1 sample (I will suggest a
volume). Vortex gently and return to the instrument. Allow the measurements to continue
for 10 minutes. If the anisotropy has not decreased by at least one third, repeat with a larger
amount of the Ulp1. Maintain a record of anisotropy vs. time.
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Plotting activity
Because the concentration of the substrate is so low, we are going to assume that the
reaction is following second order kinetics:
v = (kcat/Km)[E]tot[S]
Because only [S] varies, the reaction will follow pseudo-first order kinetics, with the rate law:
v = kobs[S]
That means the rate of reaction will vary with the decrease in substrate concentration and
the first order integrated rate law will hold:
[S]t /[S]o = e-kobs•t
Where [S]t- is the amount of substrate present at any given time and [S]o is the original
concentration of substrate. Since the anisotropy of the sample is proportional to the relative
amounts of cleaved and uncleaved substrate, the following relationship holds.
(r – rmin)/(rmax – rmin) = (r - rmin)/∆r = e-kobs•t
Or:
r = ∆r• e-kobs•t + rmin
Where r is the measured anisotropy, rmin is the anisotropy of the digested peptide and rmax is
the anisotropy of the intact substrate. You can use “R” to fit the data to this equation and
obtain a value for kobs. Your data set will involve measurements of anisotropy vs. time.
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Calculating and reporting activity
The activity of an enzyme is typically given in µmol substrate converted per minute per mg
of protein under the assay conditions. That is, in turn, obtained by taking the initial rate of
the reaction (in µmol/min) and dividing by the mass of protease added. This will take a little
bit of unit dexterity but is the essential measure of the quality of an enzyme preparation. Be
sure to include these calculations in your notebook.
Determination of Purity
The most common means of determining the purity of a protein preparation is denaturing
polyacrylamide gel electrophoresis (PAGE). Proteins are denatured by heating them with SDS, an
anionic detergent, and then applying them to the gel, which is composed of a hydrated, cross-linked
polymer. By applying a voltage across the gel, anionic molecules (such as those coated with SDS)
migrate to the anode at the bottom of the gel and separate according to size, with small proteins
migrating fast than large proteins.
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Preparing protein samples for PAGE
In consultation with me, select fractions for analysis by PAGE. Include the samples obtained
with the SMT3 fusion protein above and include samples from the crude lysate before and
after centrifugation. From each, mix 25 µL of protein solution and 25 µL of 2x SDS loading
buffer. Heat in a boiling water bath for 2 minutes. These samples may be stored
indefinitely.
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Running the gel
Load one sample per lane, and don’t forget to include a MW standard on the gel (5 µL of the
standard is sufficient). Voltage should be set to 200 V.
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Visualizing the gel
Instructions for staining, destaining and documenting the gel will be provided in lab.
Quantifying the Protein
Determining protein concentration is essential to evaluating the quality of the preparation. We will
use the Pierce BCA kit.
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Initial estimation of protein concentration
You will need to obtain a protein concentration measurement for your purified enzyme.
Ideally, the protein should be between 0.2 to 1.0 mg/mL, but we don’t know the
concentration. I suggest using the protein in three concentrations: (i) as it was from dialysis,
(ii) diluted 5x and (iii) diluted 25x. You will need 100 µL of each of the three concentrations.
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Prepare standards
A standard sample of bovine serum albumin (2 mg/mL) will be provided. Prepare a series
of five dilutions from 0 to 1.0 mg/mL, inclusive. You will need 200 µL of each.
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Perform assay
Prepare 20 mL of working reagent by mixing 50 parts of reagent A with 1 part of reagent B.
In a series of labeled microcentrifuge tubes, add 50 µL of protein solution (either standard or
Ulp1 sample) and 950 µL of working solution. Prepare duplicates of each standard and
unknown solution for a total of 16 samples. Do this in a timely fashion – there should be no
more than a five minute period from beginning to end. Place samples in a 37˚C water bath
for 30 minutes. Then remove, cool to RT in a water bath and measure absorbance at 562
nm. All absorbance readings should be collected within a 10 minute period.
The Write-up
Abstract:
As always, briefly summarize purpose of the work and the results. In this instance, some mention of
quantity of protein (obtained from 1 L of bacterial culture), purity and activity.
Introduction:
Brief description of the protein being purified. I recommend the article from Lima’s lab (posted on
the Moodle), which describes the proteins and provides some helpful refs.
Experimental:
Several techniques are engaged in this experiment: purification & dialysis, quantitation, SDS PAGE,
and activity assay. Each will require a short but authoritative description. I have placed a copy of
the experimental section of Kristin Grauer-Gray’s thesis (2007) for your reference, just to see how
some of these issues are detailed. It is not that close in content to what you’re doing, but gives a
sense of the appropriate presentation.
Results and Discussion:
The text need not be too extensive, but it should be accompanied by at least two figures (it will be
most convenient to print these on separate pages at the end of your document):
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Picture of the gel, with lanes clearly identified by in the image and in the caption with
contents of each lane (an example is presented on the web page).
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Decay curve, with exponential fit, for the reaction of your substrate with TEV protease.