Expression and Purification of recombinant Green
Fluorescent Protein from E. Coli strain BL21<DE3.pLysS by
Ni2+ Agarose Affinity Column Chromotography
Bibiana Enriquez
BIO 3380.102
November 11, 2013
Partner- Nirja Dalal
Graduate TA- Chantaya Konduri
Abstract
The purpose of this experiment is to express rGFP in E. Coli strain
BL21<DE3>pLysS. Throughout the experiment rGFP E. coli strain was purified using
Ni2+ Agarose Affinity Chromatography and analyzed for the amount of rGFP yield
after collecting several washes and elutions. Protein concentration of these rGFP
fractions was found by using a Bradford Assay, purity was estimated using a Sodium
Disulfide Polyacrylamide Gel Electrophoresis and detection was confirmed by
performing a Western Blot. The amount of protein concentration found in a 500ul
fraction is 21.18ug, the specific activity was found to be 251537.1901 RFU/mg and
the purity was determined to be 40%.
Introduction
Osamu Shimomura originally discovered GFP, or Green Fluorescent Protein, in 1961
from the isolation of the protein from the cnidarian, Aequorea Victoria (Prasher).
GFPs are known for their fluorescent capabilities from the excitation caused by a
photoprotein. The initial transgenic organism used to determine the specific
excitation and emission spectra was the E. Coli strain BL21 (DE3) <LysS> (Chalfie).
The emission wavelength is 510nm and the excitation wavelength is 395nm.
(Rippel) They were able to calculate the molecular weight of GFP when they realized
that GFP contained an ORF of 238 AA protein (Prasher). This revealed that there
were 238 total proteins so they multiplied the individual molecular weights by the
number of amino acids (Prasher).
In the following experiment rGFP, which is a recombinant version of GFP, was used
instead of GFP to allow for monitoring and better analysis. rGFP used contained a
His6 tag which was necessary in the purification by Ni2+ agarose column. By this
method of purification, the negatively charged histidine in the His6 tag binds to the
Ni2+ in the column. Then rGFP can be released by using an elution buffer containing
imidazole, which competes for binding to the Ni2+ because of its similarity in
structure to histidine. Since the imidazole is now bound to the Ni2+, rGFP can now
be released and collected as samples for further analysis (Rippel)
The purpose of this experiment was to express and purify a His6 tagged rGFP from
the E. Coli strain BL21(DE3)<pLysS><pRSETA-GFPuv> by utilizing Ni2+ agarose
affinity chromatography.
Materials and Methods
Expressing rGFP in E.coli
Two bacterial cultures, BL21<DE3>pLysS and pRSETA-GFPUV were cultivated in
10ml of LB growth media [100ug/ml Amp;25ug/ml Cam in 1 liter baffled flask, prewarmed-30 degrees Celsius]. 500ml of this liquid LB growth media was then
inoculated with about 4ml of overnight culture until the culture had an OD600 of 0.1
and then grown at 37 degrees Celsius with vigorous shaking until it had an OD600 of
0.5. After taking 1ml of the culture and pelleting in a 1.5ml centrifuge tube, the
supernatant was discarded while the pellet was labeled G0 and stored at -20
degrees Celsius. The culture was induced with a final 1mM concentration of IPTG. 3
hours post induction, 1ml of the culture was pelleted in a 1.5ml centrifuge tube,
labeled G3 and stored at -20 degrees Celsius. 15ml more of the culture was pelleted
in a 15ml centrifuge tube, labeled G3-15ml and stored at -20 degrees Celsius.
Preparing the GCE
When slowly freezing the bacteria, small ice crystals were formed so that the cell
wall of the bacteria could be damaged and the lysozyme from cytoplasm could be
released into the breaking buffer and would be allowed to damage the cell wall of
other surrounding bacteria. This is helped by vortexing. 1ml total of breaking buffer
[10mM Tris, pH 8; 150mM NaCl] was added to the bacterial pellet and was then
thawed and homogenized by pipetting up and down. This was then vortexed,
labeled and placed in a 37 degrees Celsius water bath for 10 minutes. This was then
put in a platform shaker in a dry air 37 degrees Celsius incubator for 20 minutes to
keep lysing. The mixture was centrifuged at 14,000xg, 4 degrees Celsius for 10
minutes.
Ni2+ -NTA Chromatography Separation
A small, 3ml plastic syringe packed to about the 1ml mark with glass wool was used
as a filter. 1ml of breaking buffer was pipetted into the syringe to prevent air
bubbles from remaining in column. The breaking buffer was allowed to drip out so
the leur-lock could be secured on without risk of getting air bubbles. .5mL bed
volume of Ni2+ -agarose was added to the column with the leur-lock open. 5ml of
breaking buffer, which was used as a washing buffer, was added to the column to
take out ethanol from agarose. Crude extract was then added. After 5-10minutes the
leur-lock was opened and the liquid from column was allowed to drip so that
washes could be collected in increments of 0.5ml. These washes were labeled “W3W10”. Elution buffer was then added in increments of 0.5ml to allow collection of
our 10 elutions, which were labeled E1-E10.
Protein Concentration Calculation using Bradford Assay
First, a standard curve was developed using the following known concentrations of
BSA protein: 0ug, 2.5ug, 5ug, 10ug, 15ug and 20ug. Bradford reagent was added to
these samples and the volume was brought to 50ul by adding water until it reached
the desired 50ul. The known samples were then read in a microplate reader at an
absorbance of 595nm to determine their individual absorbances. These absorbances
were then plotted against the known concentrations to serve as a guide for
extrapolating data after determining a best-fit line. The purified samples with
unknown concentrations then underwent a similar Bradford assay in triplicate. The
final total protein amount was then calculated by extrapolating the value from the
standard curve developed previously.
SDS-PAGE Analysis and Purity Determination
First the SDS-PAGE is made using a 12% Resolving gel containing 4X resolving
buffer (0.75M tris pH 8.8, 0.4% SDS), water, 30% Acrylamide, 10% APS and TEMED
needs to be poured and polymerized. Then the 5% stacking gel containing 4X
stacking buffer instead of resolving buffer was also poured and allowed to
polymerize. Exact quantities used can be found in Rippel’s biochemistry laboratory
manual referenced below. The polymerized gel was then placed inside the
electrophoresis tank and the rGFP samples were loaded. The samples used were G0,
G3, GCE, W4,W5,E4, and E5. A ladder was also included so that it could be used as
reference for the molecular weight. Electrophoresis was then conducted under 200
volts for about 45 minutes. Then Coomassie Blue was used to stain the gels so
visualization of bands would be possible.
Western Blot and Protein Presence Verification
An SDS-PAGE gel was prepared and the samples had B-mercaptoethanol added
before loading into the gel. The protein bands were then transferred onto the
nitrocellulose membrane. Then Ponceau S dye was used to stain the membrane for
about a minute and then deionized water was used to rinse which allowed the bands
to become visible. The membrane was then blocked by incubating in 5% nonfat dry
milk and then washed with 0.05% Tween 20/TBS. Mouse IgG anti-Xpress epitope
MAb solution was added to the membrane and then washed again. Sheep IgG antimouse IgG conjugated horse-radish peroxidase polyclonal anti-serum is then added
and the membrane is washed twice more. TBS is then used for the final wash. The
Western Blot is developed by adding TMB solution and then stopping the reaction
by placing the membrane in distilled water.
Results
The G0 sample from the G strain BL21 (DE3) <pLysS, pRSETA-GFPuv means that it
was taken at time equals 0, which implies that no induction has taken place.
Similarly, G3 is the sample that was taken after 3 hours post induction by IPTG.
Inhibition of the Lac Repressor by IPTG induces T7 RNA polymerase which binds to
the T7 promoter causing transcription of the rGFP gene and therefore allowing rGFP
mRNA to be made. After translation, the rGFP protein is formed. Also in the plasmid
is ampicillin resistance which is necessary because ampicillin was used to help
maintain selection and prevent contamination from other bacteria. (Rippel)
PT7
ATG-His6-Xpress-GFPUV
pRSETA-GFPUV
Figure 1
Plasmid Map of rGFP
The figure above is the rGFPUV plasmid. There is a Pt7 promoter, which initiates
mRNA transcription. His6 and Xpress are tags. If the inducer, IPTG is present, the t7
promoter binds to t7 RNA polymerase and rGFP is encoded. The ampicillin
resistance is there to maintain selection for the plasmid so plasmids are not lost.
Schematic Diagram of rGFP
Xpress Epitope Tag
His6 tag
1
5
5’ N terminus
GFPUV
11 24
32
chromophore
GFPUV
39 104-106
277
279
3’ C Terminus
Figure 2
This is a schematic diagram of rGFP. Labeled first is the 5’ and 3’ ends of the
sequence. Labeled next is the His6 tag which is 6 His molecules transcribed in the
“CAT” sequences. The next tag seen is the Xpress Epitope Tag starting at the “GAT”
and ending at the “AAG” codon. The actual GFPUV DNA sequence is 238 amino acids
long and is towards the 3’ end. The chromophore is located at the 65-67 amino acids
of GFP and is what is responsible for the fluorescence of GFPuv .
Figure 3
rGFP Activity and amount in ug after Purification using Ni2+ agarose
rGFP in the E. Coli strain BL21(DE3)<pLysS><pRSETA-GFPuv> was purified through
a Ni2+ agarose column with a bed volume of 0.5mL. 10 washes and elutions were
collected afterwards, using first a washing buffer, then elution buffer. rGFP amount
was found by performing a Bradford assay in triplicate. The results for the first 6
washes and elutions are shown below.
SDS-PAGE Gel of rGFP samples (G0, G3,GCE, W4,W5,E4,E5, ladder) using 12%
Resolving Gel and 5% Stacking Gel and stained with Coomassie Blue
Figure 4
Above is an SDS-PAGE gel resulting from a 12% Resolving gel (4X resolving buffer
(.25M tris pH 8, 0.4% SDS), 30% Acrylamide (29.2% w/v acrylamide, 0.8% w/v bis
acrylamide), 10% APS, TEMED) and a 5% stacking gel (4X stacking buffer (.25M trus
pH 6.8, 0.4% SDS), 30% Acrylamide (29.2% w/v acrylamide, 0.8% w/v bis
acrylamide), 10% APS, TEMED. The 15ul samples consist of previously purified
rGFP fractions of E. coli strain BL21 <DE3>pLysS, pRSETA-GFPuv by Ni2+ agarose
column.
Western Blot
GO
G3
GCE
W4 W5 E4
E5
LADDER
97.4kDa
66.2kDa
45kDa
31kDa
21.5kDa
14.4kDa
Figure 5
An SDS-PAGE gel was prepared with the same samples as with the SDS-PAGE
analysis but had 2-ME added to each. The gel was run at 500V. The samples and
ladder were loaded as labeled above. The first column does not show any bands
because it represents G0. The following columns had the strongest band at about
35kDa. G3 and GCE had an excess of bands because too much sample was loaded in
gel. The bands present at around 35kDa validate the presence of rGFP because its
molecular weight is close to that value.
Conclusion
In this experiment W5 and E5 had the highest fluorescence, which meant that it also
had the greatest amount of protein and specific activity. This was confirmed with
the calculation of specific activity, which is RFU/ mg of protein. The average
molecular weight of rGFP was calculated to be 33.12kDa on the basis of the number
of amino acids in rGFP and the weight of each individual one. The estimated
molecular weight using SDS-PAGE was about 35kDa, which is close to the calculated
value. The purity was found to be 40%.
Scientists can further use GFP to monitor bacteria in animals to pinpoint the
location of bacteria based illnesses in the case that other tests were inconclusive or
more intrusive. More experiments can be conduced to determine how GFP reacts to
the conditions, such as internal body temperatures, in animals to see if there are
certain species that would adhere to this procedure better than others.
References
Chalfie, Martin. “Green Fluorescent Protein as a Marker for Gene Expression.”
ddddScience. Feb (1994): 802-05.
Prasher, Douglas C. “Primary structure of the Aequorea victoria green-fluorescent
ddddprotein.” Gene 111.215 Feb. (1992): 229-33
Rippel, Scott. Biochemistry Lab Manual and Lecture Notes. 2013.