BIOTECHNOLOGY I – PROTEIN PURIFICATION BY ION EXCHANGE Written by Eilene Lyons Revised 2/25/10 4-1 LAB 4 PROTEIN PURIFICATION BY ION EXCHANGE STUDENT GUIDE GOAL The goal of this laboratory is to teach the basics of ion exchange chromatography, DNA restriction, and specific activity of enzymes. OBJECTIVES After this lab, the student will be able to 1. Describe the molecular action of Type II endonucleases. 2. Explain why cellular DNA is not degraded by the cell’s endonucleases. 3. Set up and use a chromatography column to purify a protein from cell extract. 4. Assay fractions from column chromatography of bacterial cells for DNA restriction enzyme concentration 5. Calculate the units of activity and the specific activity of an enzyme from experimental results. 6. Describe the difference between total activity and specific activity of an enzyme. 7. Apply knowledge of unit activity and specific activity when ordering enzymes. TIMELINE Day 1 – Prep, collect column fractions, cast agarose gel Day 2 – First Assay Day 3 – Second Assay and data analysis BACKGROUND Restriction Enzymes Bacteria use restriction enzymes to destroy invading virus DNA by cutting it into fragments along the sugar-phosphate backbone of the DNA. Immediately after DNA replication, bacteria protect their own DNA by adding methyl groups to restriction recognition sites recognized by their own restriction enzymes, preventing binding of these enzymes to the cellular DNA. Some restriction enzymes cut from a free end of the DNA (exonuclease), while others cut along the backbone (endonuclease). Since Type II endonucleases cut at sequence-specific sites and are the type of enzyme used most often in genetic engineering, they are referred to simply as restriction enzymes in the laboratory. Over 1500 different restriction enzymes have been found since the first was discovered by H. O. Smith and colleagues in 1968. Most restriction enzymes are either protein dimmers (two subunits of equal mass between 20,000 and 25,000 daltons) or single polypeptides of molecular weights from 30,000 to 35,000 daltons. Different restriction enzymes use different recognition sequences (usually palindromes) of DNA where they attach and cut. A palindrome is a word or phrase spelled exactly the same forward and backward. DNA palindromes involve both strands of the molecule, where the sequence on the bottom strand is the opposite of the complementary base pair sequence on the top. For example, GAATTC CTTAAG
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BIOTECHNOLOGY I – PROTEIN PURIFICATION BY ION EXCHANGE
Written by Eilene Lyons Revised 2/25/10 4-1
LAB 4
PROTEIN PURIFICATION BY ION EXCHANGE
STUDENT GUIDE
GOAL
The goal of this laboratory is to teach the basics of ion exchange chromatography, DNA
restriction, and specific activity of enzymes.
OBJECTIVES
After this lab, the student will be able to
1. Describe the molecular action of Type II endonucleases.
2. Explain why cellular DNA is not degraded by the cell’s endonucleases.
3. Set up and use a chromatography column to purify a protein from cell extract.
4. Assay fractions from column chromatography of bacterial cells for DNA restriction enzyme
concentration
5. Calculate the units of activity and the specific activity of an enzyme from experimental
results.
6. Describe the difference between total activity and specific activity of an enzyme.
7. Apply knowledge of unit activity and specific activity when ordering enzymes.
TIMELINE
Day 1 – Prep, collect column fractions, cast agarose gel
Day 2 – First Assay
Day 3 – Second Assay and data analysis
BACKGROUND
Restriction Enzymes
Bacteria use restriction enzymes to destroy invading virus DNA by cutting it into fragments along
the sugar-phosphate backbone of the DNA. Immediately after DNA replication, bacteria protect
their own DNA by adding methyl groups to restriction recognition sites recognized by their own
restriction enzymes, preventing binding of these enzymes to the cellular DNA. Some restriction
enzymes cut from a free end of the DNA (exonuclease), while others cut along the backbone
(endonuclease). Since Type II endonucleases cut at sequence-specific sites and are the type of
enzyme used most often in genetic engineering, they are referred to simply as restriction
enzymes in the laboratory. Over 1500 different restriction enzymes have been found since the
first was discovered by H. O. Smith and colleagues in 1968. Most restriction enzymes are either
protein dimmers (two subunits of equal mass between 20,000 and 25,000 daltons) or single
polypeptides of molecular weights from 30,000 to 35,000 daltons. Different restriction enzymes
use different recognition sequences (usually palindromes) of DNA where they attach and cut. A
palindrome is a word or phrase spelled exactly the same forward and backward. DNA
palindromes involve both strands of the molecule, where the sequence on the bottom strand is the
opposite of the complementary base pair sequence on the top. For example,
GAATTC
CTTAAG
BIOTECHNOLOGY I – PROTEIN PURIFICATION BY ION EXCHANGE
Written by Eilene Lyons Revised 2/25/10 4-2
Different species have different restriction enzymes, so each has been named according
to the species where they were discovered. The Roman numeral denotes if the enzyme
was the first, second, third, etc., enzyme found in the species. Some restriction enzymes
recognize the same DNA sequence and are referred to as “isoschizomers.” The restriction
enzymes Cla I and Bsp 106 both attach to the DNA sequence ATCGAT and cut between
the T and C. Molecular biology product catalogues give isoschizomer lists and sequence
recognition sites for most enzymes used in the research laboratory. There are reports in
the scientific literature of restriction enzymes cutting at more than one recognition site
due to changes in the cation concentration, the pH of the solution, or the presence of
glycerol in the reaction mixture. This indiscriminate cutting is referred to as star activity,
and can also be caused when the restriction enzyme concentration is too high.
Some endonucleases, like EcoR V, cut leaving blunt ends. Others cut asymmetrically,
leaving cohesive or sticky ends – nucleotides that are not hydrogen bonded to other
complementary nucleotides. Once cut from within, DNA polymers separate as the
hydrogen bonds between the cuts break. See Table 1.
5’ CGAATG AATTCTACCCAAG 3’
3’ GCTTACTTAAG ATGGGTTC 5’
Table 1. Examples of Restriction Endonuclease Recognition And
Cutting Sites ( and spaces denote where the DNA backbone is cut.)
EcoR I: G AATTC G GATCC BamH I:
Escherichia coli, CTTAA G CCTAG G Bacillus amyloliquefaciens H
strain RY 13
Hind III: A AGCTT GAT ATC EcoR V (a blunt cutter):
Haemophilus TTCGA A CTA TAG Escherichia coli,
influenzae Rd strain RY 13
PRACTICE: Identify the restriction site in each DNA sequence below. Write the name of the
enzyme on the line beside each sequence. Mark the cutting sites with slashes. Draw a horizontal
line showing what hydrogen bonds will break, separating the two polymers of DNA.
BIOTECHNOLOGY I – PROTEIN PURIFICATION BY ION EXCHANGE
Written by Eilene Lyons Revised 2/25/10 4-3
Restriction of Lambda DNA by Eco RI
The most widely used method for determination of a restriction enzyme’s cutting ability
is to assay it using DNA from a small viral genome and running the resulting fragments
on an agarose gel for analysis. To assay for Eco RI activity in this lab, a sample of each
fraction collected by column chromatography will be incubated with chromosomal
Lambda viral DNA to see which of the fractions cuts the DNA. To determine cutting, the
DNA restrictions digestions will be applied to an agarose gel to determine if all the
known Eco RI sites are completely cut, giving an expected number of fragments on the
gel. There are 5 Eco RI sites in the Lambda chromosome, giving linear fragments of the
following sizes when cut: 21226, 7421, 5804, 5643, 4878, 3530 bp. The two fragments of
5804 and 5643 bp are so similar in size that there may not be separation to show two
distinct bands if the gel is not run long enough. Therefore, you may see either 5 or 6
bands on the gel, depending on how long it was electrophoresed and whether there was
enough Eco RI present to cut the DNA completely.
Ion Exchange Chromatography
Purification of restriction enzymes can be accomplished using Ion Exchange
Chromatography. In column chromatography, a resin or column packing compound, such
as polystyrene or a polysaccharide such as Sepharose, Sephadex, or Diethylaminoethyl-
Cellulose (DEAE-Cellulose), is “packed” into a column. The resin must be kept moist
with a buffer that covers it so that it does not dry out, preventing it from functioning
properly. The sample containing the protein of interest is applied to the top of the column
and allowed to enter the column as drops of the buffer are collected off the bottom (called
“fractions”). See Figure 1. The proteins in the sample bind to the charged ions of the
column, and the column is washed with buffer at a pH and salt concentration that
maintains the interaction of the proteins with the resin. The salt concentration of wash
buffer is gradually increased so that only the protein of interest binds tightly. As the salt
concentration is increased further, the protein of interest releases (elutes) from the resin
and will drip through the column. Measured fractions of wash and elution buffer are
collected and then tested for desired enzyme activity.
Figure 1. Steps of Ion Exchange Chromatography
Bound protein of interest
Other proteins
BIOTECHNOLOGY I – PROTEIN PURIFICATION BY ION EXCHANGE
Written by Eilene Lyons Revised 2/25/10 4-4
Calculating Total Activity The activity of an enzyme can be measure in two ways, as units of activity or as specific
activity. A Unit of activity is defined as the amount of enzyme that fully cuts a given
amount of substrate in a given amount of time under standard assay conditions. For
example, if the assay conditions are that 1 µg of DNA is used and that the reaction is to
be incubated for 1 hour, resulting in complete cutting of the DNA as determined by gel
electrophoresis, then the enzyme volume used is equivalent to 1 Unit of activity. See the
sample problem, below.
Calculating Specific Activity
Specific activity is the number of Units of activity per milligram of protein present in the
sample. Specific activity is a way of saying how much of the protein in the fraction is the
enzyme of interest, in this case, EcoR I. In industry, specific activity is a measure of
purity - how much enzyme there is per the total protein in the sample. This has a bearing
on how to select enzymes for purchase. You may find several forms of an enzyme from a
chemical supplier, some containing more Units of activity, while others show higher
specific activity. The amount of purity may be more important than the cost or more
important than how much you must use for your application, in which case, you would
opt to pay more and get the more highly purified enzyme rather than the cheaper, less
purified one. To calculate the specific activity of the enzyme from the sample problem,
above, you would first need to take a UV spectrophotometer absorbance reading to get
the milligrams of protein per milliliter. Suppose that the A280 was 0.234. Recall that the
spec reading at 280 nm is the milligrams of protein per milliliter that are present in a
sample. The calculation of specific activity would then be:
Sample Problem: By definition, one Unit of Eco R I enzyme activity is equal to the amount of enzyme that can digest 1 µg of genomic Lambda viral DNA in 60 minutes at 37°C. Units of activity are expressed as Units per milliliter of enzyme solution. A sample of the fraction is diluted 1 to 4. Ten microliters of the dilution showed complete
digestion of 1 g of Lambda DNA in 30 minutes. The amount of enzyme in that 10 µl of fraction (we don’t know its molar amount) is equivalent, therefore, to 1 Unit of activity. Multiply by the dilution factor (4) and by 2, for how much would be cut if you incubated for twice that long (since by definition 1 unit is how much enzyme it takes to cut the DNA in one hour).
(1U/10 l) x 4 x 2 = 8U/10 l = 0.8 U/l
To calculate how much is in 1 ml of the fraction:
(0.8 U/l) x 1000 l/ml = 800 U/ml
Specific Activity = (800 U/ml) ÷ 0.234 mg/ml = 3,418 U/ mg protein
BIOTECHNOLOGY I – PROTEIN PURIFICATION BY ION EXCHANGE
Written by Eilene Lyons Revised 2/25/10 4-5
Buying Enzymes for Research
Here are some points to consider before you buy an enzyme:
1. What is the application? Will the protocol make a difference?
2. How pure does the enzyme need to be, i.e., will other contaminants have an effect
on the experiment?
3. What is the size of the reaction for which the enzyme will be used? Is it better to
use a small amount rather than a large amount of enzyme in a single reaction in
order to get the substrate completely degraded?
4. How important is the cost? Is there a minimum amount you can spend or do you
want what is best for the purpose, regardless of the price?
5. How long does the enzyme keep? If it has a long shelf life, it may be better to buy
more so that you pay less per mg.
6. How many units will be needed based on previous tests?
LAB OVERVIEW In this lab, the restriction endonuclease Eco R I will be isolated from a bacterial cell
lysate by ion exchange chromatography. The fractions collected will be assayed by
incubation with Lambda chromosomal DNA. Units of activity and specific activity of the
isolated enzyme will then be calculated.
SAFETY GUIDELINES
Good Laboratory Practice requires wearing safety glasses and gloves. Use appropriate
safety precautions during gel electrophoresis as directed previously.
MATERIALS EQUIPMENT
Per class: Chromatography columns, one per Team
E. coli RY extract, lyophilized Ring stand with clamps, one per Team
DEAE-Cellulose Ten 13 x 100 mm test tubes per Team
10x Equilibration Buffer Gel electrophoresis units, one per Team
50% Glycerol 5 ml serological pipette and pump, one per Team
KCl Power supplies, one per two Teams
Eco RI Reaction Buffer Waterbath set at 37°C (for digestions); 65°C for
electrophoresis and 1.5 ml tube floats
Molecular grade water (Qualified water) UV Spectrophotometers, two
Lambda DNA Matched Quartz Cuvettes & cuvette rack
Lambda/Eco RI Marker Automatic micropipetters and tips