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Protein expression and purification
Introduction
In
order to
produce and study our different protein constructs they had to be
expressed in
bacteria. After
cloning the individual
parts into the pMA vector the complete expression products were then
transferred
into the pEx vector (see pEx vector ) and transformed into competent E. coli expression strains via heat
shock. We used two different strains for the protein expression: E. coli BL21 de3 (Novagen) and E. coli RV308 (Maurer et
al., JMB 1980). Both strains are suited
for high-level protein expression.
The
expression level of proteins can vary in many parameters, so the
conditions
often need to be optimized after the first try. Some of these
expression
parameters are: host strain, temperature, time of induction, inducer
concentration, harvest time, etc.
Methods
After
the successful
expression of a construct, the protein of interest had to be purified
from the
cell lysate created using a sonifier or a french press. There are
different
methods to do this, one of the easiest is to fuse an affinity tag to
the protein
of interest. We used three different tags: 6x Histidine-tag ,
Streptavidine tag
and a GST-tag in different approaches. To obtain the tagged proteins a
base
sequence coding for the information for the tag amino acid sequences
has to be
inserted at the beginning or at the end of the construct. A specific
and
commercially available binding partner exists for each tag and by
exploiting
this interaction, the protein of interest can be purified. Hence in
most cases,
the protein purification starts with the loading of the cell lysate
onto a
column, which contains a matrix coupled to one specific binding
partner, such
as Ni-NTA (Qiagen Ni-NTA Superflow) for the His-Tag, StrepTactin (IBA Strep-Tactin Superflow high capacity)
for the Strep-tag and Glutathione (GE Glutathione Sepharose High
Performance)
for the GST-tag. The tagged proteins bind specifically to their binding
partner
and whereas the other, non-tagged proteins are washed away. The protein
of
interest can then be eluted with a solution containing a competitive
molecule.
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Figure:
protein purification scheme |
Problems
can occur if the protein of interest is too fragile or toxic for the
cells. To
avoid killing the cells with a toxic protein we used the method of
exporting
the protein into the periplasm of E. coli
by adding a signal sequence (of the periplasmic protein DsbA) at the
beginning
of the target protein so that it will be cotranslationally exported and
can’t
cause any damage in the cytoplasm. The signal sequence itself gets
split off by
a protease in the periplasm, hence interference with the protein is
avoided.
For
running
the column we used the AKTA 900 FPLC purifier system from Amersham
Pharmaceuticals.
The fluorescence pictures were taken with a Zeiss AxioVision
MTB2004 fluorescence microscope
with a COLIBRI LED system.
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Left
picture: Laura preparing the ÄKTA purifier; right
picture: Laura and Hannes using the Zeiss AxioVision fluorescence
microscope.
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Results and Discussion
Our
first proteins
chosen for expression were the argonaute proteins (Ago) from Aquifex
aeolicus
and Thermus
thermophilus. The Ago
protein was tagged with a polyhistidine tag so it could be
purified by using a Ni-NTA column. We transformed the vector with the
Ago
sequence into both E. coli BL21de3
and E. coli T7 Express (NEB) which
is
an enhanced derivative of the BL21 strain. SDS gel electrophoresis
showed that
both strains expressed the Ago protein at a high level. The cells were
harvested after 8 hours of growth at 20°C. The cell pellets
were resuspended in
washing buffer and a proteinase inhibitor cocktail was added before the
cell
suspension was sonicated and filtered. Then the filtered cell lysate
was loaded
on the Ni-NTA column, washed and eluted with a buffer containing 250mM
imidazole. The elution fractions were subsequently mixed with Laemmli
buffer
and loaded on a SDS gel and stained with Coomassie.
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SDS
gel ; argonaute protein from
Aquifex aeolicus;
lanes: Marker (NEB prestained protein marker broad range), elution
fraction 1,
elution fraction 2, elution fraction 3, elution fraction 4, elution
fraction 5,
elution fraction 6, elution fraction 7, elution fraction 8 |
The size of the Ago protein is about 83 kDa and distinct bands can be seen. Fractions 1-4 were pooled and further purified by size exclusion chromatography with a GE HiPrep 16/60 Sephacryl S-200 HR column.
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SDS
gel ; argonaute protein from Aquifex
aeolicus
after Ni-NTA column and size exclusion chromatography;
lanes:
Marker (NEB prestained protein marker broad range), fraction 1 (after
size
exclusion), fraction 2, fraction 3, fraction 4, fraction 5, fraction 6,
fraction 7, fraction 8, fraction 22 |
In
lane 10 a
distinct band is visible and the amount of unwanted proteins is much
lower.
Our
second
project was the purification of a Fok construct. We started with the
expression
of an inactive Fok construct (His-Flu_A-SplitLi-Fok_i in the pEx
vector). The first
Fok_i construct was expressed in
BL21 de3 at 24°C. The cells were harvested after 4 hours of
growth.
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SDS
gel; pEx His-Flu_A-SplitLi-Fok_i in BL21de3 ;
Lanes: Marker (NEB prestained protein marker broad range), elution
fraction 1, elution
fraction 2, elution fraction 3, elution fraction 4, elution fraction 5,
flow
through fraction 1, flow through fraction 3, washing fraction 1 (250mM
imidazole), washing fraction 3 |
The results of this expression were also verified with a Western Blot using antibodies directed against the terminal His-tag and three positive controls were included.
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Western
Blot; pEx His-Flu_A-SplitLi-Fok_i in BL21de3;
Lanes: 1. Marker (NEB prestained protein marker broad range), 2. sample
of pool
(fractions 1-4), 3. empty lane, 4.-6. 3 positive controls
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The
hardest
step was the expression of a construct containing the active Fok
protein (His-Dig-Split-Fok_a
in the pEx vector). After several failed attempts to express the Fok_a
protein
and purifying the protein of interest out of the cell lysate with a
Ni-NTA
column we tried to tag the protein with a Strep tag
(Strep-DigA-Split-Fok_a).
We made several attempts with variations in the growth temperature, the
time of
induction (at higher OD) and the amount of IPTG. However, in all cases
the
cells stopped to grow about 2 hours after induction. So in the next
attempt we added
a signal sequence for periplasmic export to the protein
(DsbA-His-DigA-Split-Fok_a).
By exporting the protein into the periplasm its toxicity can be reduced
and the
purity of the protein gained by export in the periplasm is usually
higher as
well. We performed several attempts to purify the Fok_a construct in
this setup
but did not obtain detectable amounts of protein. Our next attempts
were to add
a His-tag in addition to the Strep-tag to purify even small amounts of
the
Fok_a construct because the expression level remained low and the
protein still
proved toxic for the cells. A second attempt for the purification of
small
amounts of an active Fok fusion protein was to use a Glutathione column
which
was loaded with a GST-tagged FosW, which is a specific Fos-binding
helix
selected by phage display. We then created a construct containing a Fos
linked
to an active Fok (His-Fos-SplitLi-Fok_a), so that
the GST-linked FosW binds to the Glutathione
immobilized on the
column can be bound
by FosW.
Since
several approaches to detect and to purify active Fok proteins failed,
we also
added a fluorescent protein (Venus) to the protein construct. We
detected a
strong fluorescence under the fluorescence microscope after induction
of the
cells. The protein construct can be purified using a GFP-trap column.
.JPG) |
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Picture
taken with a fluorescence microscope using a YFP
filter; Fok_a_Venus expressed in BL21 cells
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The Venus protein was fusioned downstream to the protein construct, so the protein construct is expressed in any case when a fluorescence signal of the Venus protein can be seen.
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SDS
gel; pEx Venus-Fok_a in BL21de3; lanes: 1. Marker
(ColorPlus Prestained Protein Marker, Broad Range), 2. clone 1
uninduced, 3. clone
1 induced, 4. clone 1 induced, 5. clone 2 uninduced, 6. clone 2
induced, 7. control
(His-FluA-SplitLi-Fok_i + His-DigA-SplitLi-Fok_a) uninduced, 8. control
(His-FluA-SplitLi-Fok_i
+ His-DigA-SplitLi-Fok_a) induced
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In
the
lanes of the induced clones ( lanes 3,4) appears a band at about 50 kDA
which
could be the Venus-Fok_a construct.
To
detect
whether the protein construct with the linked GFP is completely
expressed or
gets degraded we did a western blot with specific anti-GFP antibodies
(first
antibody: Santa Cruz Biotechnology sc-9996 mouse αGFP; second
antibody: Santa
Cruz Biotechnology sc-2005 goat αmouse IgG-HRP)
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Western
Blot; pEx Venus-Fok_a in BL21de3; lanes: 1. Marker
(NEB prestained protein marker broad range, 2. clone 1 uninduced, 3.
clone 1
induced, clone 1 induced, clone 2 uninduced, clone 2 induced, control
(His-FluA-SplitLi-Fok_i
+ His-DigA-SplitLi-Fok_a) uninduced, control (His-FluA-SplitLi-Fok_i +
His-DigA-SplitLi-Fok_a) induced |
All
of the
induced clones show a strong signal but show many different bands on
the
membrane. There’s a signal at 49 kDa, which is the size of
the Venus-Fok_a
complex. The bands indicating proteins of smaller size could be
truncated
proteins.
Due
to the
lack of time at the end our assays to purify Fok_a proteins are still
under
progress and we keep trying to reach successful expression and
purification.
