Name: Axel Blom

Student number: s1518410

Room: 7
Date: 15 january 2015

Name partner: Kasper Bergsma

Assistents: Max Clabbers, Soedish Sardjoepersad, Raffaella
Tassoni and Lieke van den Eijnden

Protein purification: Isolation and

characterization of His6--galactosidase

Abstract
The purpose of this experiment was to isolate and purify His6--galactosidase from an E. Coli strain
and confirm both the isolation and purification. The isolation was done through lysis of the cells,
isolation of soluble protein and elution with a stationary phase of cobalt ions binding to the histidine
residues. The protein was then slowly removed from the stationary phase using a linearly increasing
imidazole gradient. Of the elution, the three peak fractions were taken as product.
The product was analyzed by comparison with samples taken during the purification process. The
methods of analysis were SDS-PAGE, native PAGE and absorbance measurements to obtain the
activity and quantity of the protein. The results of these analyses are that the product has a
purification factor of about 1.5 and a yield of about 13% of the original activity was achieved. The
purification was therefore successful, although other methods should be considered to obtain large
quantities of protein.

Introduction
Enzymes are biological catalytic molecules used by every living organism to perform otherwise
impossible chemical reactions. Notable traits of enzymes are an incredible specificity of reaction
catalysis and how they are affected by the concentration of both substrate and product. These traits
make enzymes well worth studying. To be able to study these proteins, however, they must first be
obtained. This can be done by utilizing the specific traits of the protein, such as mass, charge and
binding of a substrate. The protein can also be chemically altered to interact with specific substances
to separate it from all the other proteins. An example of this is the addition of a histidine tag, a set of
6 extra histidines at the N-terminus of a protein.
The purpose of this experiment is to obtain -galactosidase by extraction of the protein from E. Coli.
and purification by means of elution. -galactosidase is an enzyme used by the bacterium to utilize
lactose as an alternate source of energy. To obtain the protein, cells which have been overproducing
-galactosidase are lysed, treated with several other chemicals and centrifuged a number of times
until nearly all that remains is protein. This protein solution is then eluted in a buffer with a cobalt
ion surface as the stationary phase. The histidine-tag will bind -galactosidase to the ions, but all the
other proteins will be washed away. Then an increasing imidazole gradient is used to slowly release
the -galactosidase from the stationary phase. The resulting product is then analysed.
The product and samples taken during the purification process are analysed through SDS-PAGE,
protein electrophoresis based purely on protein mass, native PAGE, protein electrophoresis based on
specific charge and mass, and through activity measurements with ONPG. The PAGEs are performed
using discontinuous gels: they consist of a stacking layer and a separation layer. In the top stacking
layer the proteins travel fast so that when they reach the separation layer they are squashed into
condensed lines. The SDS gel is coloured with a generic protein colouring, but the native gel is
coloured using X-gal, which forms a bright blue substrate after treatment with -galactosidase . The
activity is measured using the absorbance over time of ONP, the substrate of ONPG and galactosidase, which has a bright yellow colour. The amount of protein is measured via a standard
Bradford assay.

Materials and methods

The complete method used is stated in the practical course manual Life Sciences 2014-2015 (De Smit
& Vijgenboom, 2014). There are, however, some notable facts and deviations from protocol. A
devation from protocol was using twice as much buffer A to wash the column by hand after the
column fell dry to make sure it was washed out properly.
Buffer A: 117 ml 0.2M Na2HPO4, 8 ml 0,2M NaH2PO4, 30 ml 5M NaCl, 345 ml H2O
Buffer B: 58.5 ml 0.2M Na2HPO4, 4 ml 0,2M NaH2PO4, 15 ml 5M NaCl, 5.617 g imidazole, HCl until pH
8, 172 ml H2O
The column material used was HisPur resin. The used fotospectrometer was number 7-6.

Results
The results of this experiment have been divided into four parts. The first part consists of a
description of the extraction of the enzyme and at which point samples were taken. The second and
third part consist of the interpretation of respectively the SDS-PAGE gel and the native PAGE gel. The
final part is an analysis of the yield and purification factor of the enzyme.

Isolation of His6--galactosidase
To extract and isolate the His6--galactosidase, an E. Coli strain mutated to produce the protein was
treated with IPTG to overproduce the protein. The resulting cell pellet was suspended in a B-PER
solution an treated with lysozyme, DNase and RNase to lyse the cell. This mixture was incubated for
15 minutes at room temperature. From this mixture, the lysate, a sample (M1) was taken. All
samples taken were put in the refrigerator immediately.
The cell suspension was centrifuged for 15 minutes. The supernatant liquid was was carefully poured
into another centrifuge tube. The pellet was used to perform a second extraction with the same
method as the first extraction. The supernatant liquid of this extraction was poured into the tube
with the other supernatant liquid. The undissolvable pellet was suspended in 1 ml of buffer A and a
sample (M2a) was taken. This mixture consists mostly of undissolvable cell structures bound to
protein aggregates. From the supernatant mixture, the dissolvable fraction, of 6.2 ml, a sample (M2b)
was also taken. His6--galactosidase is in this mixture.
His6--galactosidase was then isolated from the dissolvable fraction by means of elution. Firstly, the
dissolvable fraction was added to the column material. This was left on a shaking platform in a cold
chamber (4 C) for 60 minutes to let the histidine bind to the column material. The column material
was then poured into the column. From the liquid drained from the column a sample (M3a) was
taken. In this liquid are the remaining dissolvable proteins.
Because the column almost fell dry, it was decided to wash the column by hand (without pump).
Thereafter, the column was run with buffer A and an increasing concentration of imidazole. Every 2.5
minutes, a fraction of 5 ml was eluted. During the elution, the fractions were tested for 4

galactosidase on a 96-well microtiter plate with ONPG. From the fraction just before the fraction
where -galactosidase was eluted, a sample (M3b) was taken. The fractions with the brightest yellow
colour, fractions 11, 12 and 13 out of 40, and therefore the peak fractions were put together after a
sample(M3c, M3d, M3e) was taken from all three fractions. These peak fractions together is the
purified enzyme we will use in further experiments. These peak fractions roughly amount to an
imidazole concentration of 90 mM when most of the protein let go of the column material.

SDS-PAGE
The first method used for analysis of the product is an SDS-PAGE. In this gel, the protein is sorted
based purely on its molecular weight and coloured with the generic colouring CBB R-250. Therefore,
it is necessary to know the molecular weigth of His6--galactosidase to know where on the gel this
protein is. The MW of His6--galactosidase is 118561.07 Da (Gasteiger, 2005), so it should fall slightly
above the 100 kDa marker.
In Figure 1 it is clear to see the large bands of His6--galactosidase: it is the most notable band on the
gel and indeed slightly above the 100 kDa marker. The other large blue blurs in the lysate (lane 1),
soluble fraction (lane 3) and washed proteins from the column (lane 5) are made up of all the other
proteins from the bacteria. In the insoluble fraction (lane 2), the proteins washed from the columns
(lane 5) and the elution fraction just before the His6--galactosidase fractions (lane 6) do not show
the bright blue band
caused by His6-galactosidase. This
means that the right
products were used in
the purification process
and the right products
were discarded. The
other fractions were all
used in the purification
process.

Figuur 1. 10% SDS polyacrylamide/bisacrylamide (29:1) gel electrophoresis of several protein

samples. Lane 1: sample M1 (lysate), 1 L. Lane 2: sample M2a (insoluble fraction), 1 L. Lane 3 :
sample M2b (soluble fraction), 1 L. Lane 4: protein marker. Lane 5 : sample M3a (column washing
liquid), 10 L. Lane 6: sample M3b(no -galactosidase fraction from elution), 10 L. Lane 7: sample
M3c (first peak fraction), 10 L. Lane 8: sample M3d (second peak fraction), 10 L. Lane 9: sample
M3e (third peak fraction), 10 L. Lane 10: Prep3 (purified product), 10 L.

The peak fractions

(lane 7, 8 and 9) all
contain trace amounts
of other protein (there
are still slight blue
blurs above them), but
they are notably purer
than the first five
samples.

Native PAGE
To perform the native PAGE, the pI value of the protein must first be obtained to know what
conditions are suitable for the native PAGE. The pI value of His6--galactosidase is 5.44 (Gasteiger,
2005), so to perform the native PAGE a pH of higher than 5.44 is required. Luckily, the standard
native PAGE buffer is set at a pH of 8.3 and both the stacking and separation gel have a pH above
5.44, so there are no further measures that need to be taken to make sure the native PAGE succeeds.
The gel was coloured by laying it in an X-gal solution. When -galactosidase catalyzes the reaction to
split the X-gal molecule, the 5-bromo-4-chloro-3-indoxyl that splits of forms a dimer with a bright
blue colour. By doing this it is clear which of the sample contain the active -galactosidase. The gel is
shown in figure 2.
All of the bands in figure 2 are due to galactosidase, because that is the
enzyme that can catalyze the X-gal
reaction. All of the lanes containing
bands are samples of which we
expected there to be -galactosidase:
The lane with the lysate (lane 1) must
contain the enzyme because it contains
all the proteins of the bacterium, the
soluble protein solution must also
contain the protein because it is soluble
and all the peak fractions and the
product (lanes 6 through 9) were
already tested with ONPG. From the gel
it can also be concluded that the lysate
and soluble fraction contain a larger
Figuur 2. 5% native polyacrylamide/bisacrylamide (29:1) gel electrophoresis of
several samples. Lane 1: sample M1 (lysate), 1 L. Lane 2: sample M2a (insoluble
amount of the enzyme than the peak
fraction), 1 L. Lane 3 : sample M2b (soluble fraction), 1 L. Lane4 : sample M3a
fractions or the purified product,
(column washing liquid), 2 L. Lane 5: sample M3b(no -galactosidase fraction
because the intensity is almost the same
from elution), 10 L. Lane 6: sample M3c (first peak fraction), 10 L. Lane 7:
even though ten times as much of the
sample M3d (second peak fraction), 10 L. Lane 8: sample M3e (third peak
purified samples was used. The
fraction), 10 L. Lane 9: Prep3 (purified product), 10 L.
identification of enzymatic activity in
this gel is only qualitative. About the purity of the samples nothing can be concluded from this gel.
It was also expected that the lanes without bands were in fact without -galactosidase: The lane
containing the insoluble fraction (lane 2) should not have a band because -galactosidase is soluble
and the lanes containing elution fractions which should only contain proteins unable to attach
themselves to the column material (lane 4 and 5) also do not have bands.

Determining the yield and purification factor

The results of the Bradford assay are

shown in Table 1. The protein in the
assay was determined with a
calibration curve made in an earlier
experiment (Figure 3).

Table 1. Bradford results and determination of protein concentration.

Unit
Measured
absorbance
Protein in assay
Volume sample
in assay
Protein
concentration
in sample
Volume full
sample
Protein in full
sample

Prep1

g
mL

Prep2

Prep3

0.107
2.11

0.128
2.53

0.135
2.66

0.0002

0,0004

0.01

10550

6325

266

6.2

13.5

42.2

39.215

3.591

g/mL

mL
mg

0,6
Absorbance at 595 nm

To determine both the yield and

purification factor, several analytical
methods must be performed. In this
instance, a Bradford assay was
performed with the lysate (M1), the
soluble fraction (M2b) and the
purified enzyme (Prep3). The
reaction speed of these samples
were measured by recording the
increase of absorbance after
addition of ONP. Lastly, to make
sense of these measurements, the
extinction coefficient was
determined by averaging a plateau
of measured absorbance of a known
quantity of ONP.

0,5

y = 0,0504x + 0,0007
R = 0,9987

0,4
0,3

A595

0,2

Lineair (A595)
To determine the extinction
0,1
coefficient of ONPG, which is
necessary to calculate concentration
0
0
5
10
15
from absorbance, a known quantity of
Amount
of
protein
(g)
ONPG is added to a standard assay for
measuring extinction with a surplus of
-galactosidase. The extinction will at
Figure 3.Calibration curve for Bradford assay. This calibration curve was made by
some point plateau because all the
measuring the absorbance of known quantities of BSA.
ONPG will be converted to ONP. With
the average of the plateau and the
concentration of ONPG added, we can calculate the extinction coefficient:

0.610383(A420)
= 2298,475
4
301,25
50
To measure the activity of the enzyme, an assay was prepared with (diluted) samples, ONPG, buffer Z
and water. The assay was set for the absorbance to be read every 15 seconds for 180 seconds. The
concentration was calculated with Lambert-Beers Law:
The results are shown in figure 4.
7

ONP concentration (M)

600

M1
M2b

500

Prep3
400

Lineair (M1)
Lineair (M2b)

300

Lineair (Prep3)

200

y(M1) = 2,5187x + 29,849

R = 0,9989

100

y(M2b) = 2,8448x + 33,214

R = 0,9989

0
0

50

100

150

Time (s)

200
y(Prep3) = 2,8119x + 38,854
R = 0,9983

Figure 4. The increase of ONP concentration over time. The slope of the linear equations for the increase of ONP represent
the reaction speed of the whole reaction as catalyzed by the enzyme.

With all this data, both yield and purification factor can finally be calculated. The results are listed in
Table 2.
Table 2. Determined yield, specific activity and purification factor
Unit

Symbol and/or
calculation

Measured
reaction speed
Volume assay
Activity in assay
Volume sample
in assay
Volume full
sample
Activity in full
sample
Yield

M/s

V0

Protein in full
sample
Specific activity

Mg

Purification
factor

L
M/s
mL
mL
mol
/s
%

mol
s-1
mg-1

Vassay
Zassay= V0x Vassay
Vprep-in-assay

Prep1:
Lysate

Prep2:
Soluble
fraction

Prep3:
Purified
enzyme

2.5187
0.001
0,0025

2.8448
0.001
0.002845

2.8119
0.001
0.002812

0.0002

0.000313

0.005

6.2

13.5

60.449

56.44083

7.59213

100

93.36965

12.5596

42.2

39.215

3.591

1.4324

1.439266

2.114211

1.004768

1.475955

VPrep
Zprep= Zassayx (VPrep/
Vprep-in-assay)
(Zprep/ Zprep,
Prep1)x100
Eprep
Zsp= Zprep/ Eprep

Zsp/ Zsp, Prep1

This means that the yield of our purified product is not very high (13%) but it is about 1.5 times more
pure than the lysate. Difference between the lysate and the soluble proteins is not very big, which
means that most of the proteins in the cell are soluble.

Discussion
The purpose of this experiment was to produce, isolate and purify -galactosidase. The experiment
was successful, as a solution of purified -galactosidase was obtained through the process. This
solution can be used in further experiments concerning kinetics. From the SDS gel it can be
concluded that, although there is less of -galactosidase in the purified product, it is much purer than
the lysate and solution of soluble protein. This is confirmed by the calculations done with the activity
measurements: the final yield is low, about 13 %, but the product is about 1.5 times purer than the
lysate. In both the SDS gel and the native gel the same sample gave visible bands, except for the
sample taken from the liquid used to wash the column material. This substance did contain protein,
but not -galactosidase, which was to be expected and confirms the purification process was
performed correctly. Most of the loss of -galactosidase comes from the fact that only the three
peak fractions were taken, but almost 15 fractions contained some of enzyme.
The SDS page gel is not an ideal measure of molecular weight. Especially in this case, because the
colouring of the protein marker was not very clear, so it could only be guessed where exactly the 100
kDa marker was.
There are several steps of this experiment which did not go exactly according to plan. For example,
during the elution a tube fell out of the beaker it was in. This made the column fall basically dry,
although the problem was caught quickly. This problem occurred during the washing phase of the
elution, so no -galactosidase was lost. To solve the problem, the column was washed by hand with
about twice the amount of buffer ordinarily used. Another slight problem was the tearing of the
native gel: because it had been dry for too long, it was stuck to the glas it was poured on originally.
An assistant helped a great deal with this problem and in the end, no data was lost. The final problem
encountered was a bubble which formed in the SDS gel about 5 minutes after the gel was poured.
The bubble was in the top left corner, so after careful pouring of a small volume of the gel mixture,
the well was almost the same size as all the others.
Since about 7.5 ml column material was used and the manufacturer of the material states that 10 mg
His-tagged protein binds per ml of column material, it could hold about 75 mg. That is about 178% of
what was calculated to be in the entire lysate, so more than enough of the material was used. To
obtain large quantities of enzyme, His-tagged elution is not an ideal purification method because it is
expensive and although it provides a pure product, wastes much of the product in the process.

References
Smit, M. de & Vijgenboom, E. (2014). Practicum Life Sciences. Leiden: Universiteit Leiden
Gasteiger E., Hoogland C., Gattiker A., Duvaud S., Wilkins M.R., Appel R.D., Bairoch A. (2005).
Compute pI/Mw tool. http://web.expasy.org/cgi-bin/compute_pi/pi_tool. Visited 15-1-2015
National Center for Biotechnology Information. (2015). RecName: Full=Beta-galactosidase;
Short=Beta-gal; AltName: Full=Lactase [Escherichia coli K-12].
http://www.ncbi.nlm.nih.gov/protein/114939?report=fasta. Visited 15-1-2015