BIOL 3380- Combined Lab Report

Alicia White
Expression and Purification of recombinant Green
Fluorescent Protein (rGFP) from E. Coli using Ni2+ Agarose Affinity Chromatography

Abstract
The purpose of this experiment was to express and purify a His6-tagged recombinant
form of GFP (rGFP) from E. coli using the Ni2+ -agarose affinity chromatography. The
GFP was UV-optimized and expressed as an n-terminal His6/Xpress epitope tagged
fusion protein in the E. coli strain BL21 (DE3)<pLysS><pRSETA-GFPuv>. The crude
extract was run through a Ni2+ -Agarose column and collected in wash and elution
fractions, and then activity of rGFP was analyzed. The absorbance data from the
microplate reader found that elution three (E3) had an activity of 74157.5 RFU (Relative
Fluorescence Units). The Bradford Assay determined the protein amount of the fraction
to be 86 ug and the specific activity calculated to be 864055 RFU/mg. SDS-PAGE
estimated purity to be 55% for E3 and the yield of rGFP calculated as 47 ug. Although
the estimated molecular weight of rGFP was found to be 33.5kDa, the relative molecular
weight obtained with SDS PAGE analysis is 37kD. Western blot analysis was then done
to confirm the presence of rGFP.

Introduction
Green-fluorescent protein (GFP) was discovered by Osamu Shimomura and is used by
cnidarians as an energy-transfer acceptor in bioluminescence. This fluorescence comes
from the chemical nature of the chromophore, which is made up of modified amino acid
residues within the protein. (Prasher, Eckenrode and Ward) GFP was first cloned and
sequenced by Douglas Prasher from the cnidarian, Aequorea victoria. Wild type GFP
encodes an open reading frame (ORF) for a 238 amino acid polypeptide with a relative
molecular weight of 27kD. (Rippel) The fluorescent chromophore is stable in changes in
oxidation/reduction, chemical reagents, pH and temperature since the chromophore is
protected inside an 11 stranded Beta-barrel. (Rippel)

A recombinant version of GFP (rGFP) was expressed in E. coli, which contained a His6Xpress Epitope tag at the N-terminus. This tag was used to purify the rGFP on the Ni2+
agarose column since the negatively charged His6 tag binds to the Ni2+. An imidazole
elution buffer was used to purify rGFP because it competed for the binding of Ni2+.

The purpose of this experiment was to express and purify a His6 tagged recombinant
form of GFP from the E. coli strain BL21(DE3)<pLysS><pRSETA-GFPuv> using Ni2+
affinity chromatography technology. Once expressed and purified, the total protein
amount was estimated using the Bradford Assay and the purity, yield and relative
molecular weight were estimated using SDS-PAGE/Coomassie blue staining and western
blot was used to confirm the presence of rGFP.

Materials and Methods

Growth and Expression of rGFP in E. coli:

The G strain (BL21<DE3>pLysS, pRESTA-GFPuv) was streaked onto LB (a nutrition

rich media) plates with Ampicillin (Amp), Chloramphenicol (Cam), and IPTG. This
culture was grown overnight at 37 degree Celcius with vigorous shaking until culture was
saturated.

A sample called G0 was removed at time equals zero (OD600 ~0.5), 1ml of the culture
was pelleted and stored at -20 degrees Celcius for later. The culture was then induced
with IPTG with a final concentration of 1mM and allowed to continue growing.

3 hours post-induction with IPTG, 1ml of the culture was pelleted and this pellet is called
G3. The same was done with 15 ml of culture and this became G3-15ml. Both samples
were stored at -20 degree Celcius.

Preparing GCE:

Slow freezing at -20 degree Celcius and quick thawing was done to lyse the bacteria. 2
sets of 500 ul of breaking buffer were added into the G3-15ml pellet [breaking buffer10mM Tris, pH 8.0; 150mM NaCl]. This solution was then transferred, vortexed for 5
minutes and placed in a 37 degree Celcius water bath for 10 minutes.

The centrifuge tubes were then transferred to a rotating platform shaker in a dry air 37
degree Celcius incubator for 20 minutes to continue the lysing process. Once lysed, the
mixture was centrifuged at 14,000xg, 4 degree Celcius for 10 minutes. The supernatant
was decanted to a new centrifuge tube and labeled GCE (rGFP crude extract).

Preparing and Washing rGFP on Ni+2 agarose column:

The Ni+2 agarose column was then set up and 1ml of a 50% slurry of Ni+2 agarose was
added to the column and gravity packed. The column was the pre-equilibrated using 10fold more buffer than the bed-volume of the column.

GCE was then slowly applied to Ni+2 agarose column and 5-10 minutes were allowed to
let the histidine tag bind to the matrix. Then approximately 0.5 ml of the effluent liquid
was collected and labeled W1. Another 0.5 ml was collected and labeled W2.

The column was then further wash by adding and collecting 0.5ml increments of breaking
buffer for a total of 4ml of breaking buffer added. Each effluent was labeled W3-W10
and the column was finally washed with an additional 5ml breaking buffer.

The column was then washed with elution buffer [10mM Tris, pH 8.0; 150 mM NaCl,
300 mM imidazole] by adding and collecting 0.5ml increments to total 5ml of elution
buffer. Each 0.5ml effluent coming off the column is labeled E1-E10.

A hand held UV light was used to observe and record the qualitative fluorescence of the
W1-W6 and E1-E6 fractions.

200 ul of the W1-W6 and E1-E6 samples were loaded onto a fluorescent plate reader to
determine the quantitative fluorescence of the samples at 495nm.

Determining Protein Amount with the Bradford Assay:

A standard curve was generated with six amounts of BSA protein: 0ug, 2ug, 4ug, 6ug,
8ug and 10ug. Knowing the concentration of the BSA stock solution, 50ul solution of
protein was made with the respective amounts and 200ul of Bradford reagent was added
to the samples. The absorbance of these samples was measured at 595 nm in the
microplate reader and a standard curve of Absorbance (595 nm) vs. BSA (ug) was plotted
and a best fit line drawn.

This same procedure was then repeated in triplicate for the samples of W1-W6 and E1-E6
and the absorbance data was collected and verified against the standard curve. By
extrapolating the absorbance value on the standard curve, the total protein amount was
able to be determined in the volume of sample pipetted in the Bradford Assay for these
samples of unknown concentration.

SDS-PAGE Analysis:

The SDS-PAGE is made using a 12% resolving gel with 4x resolving buffer (0.75M tris
pH8.8, 0.4% SDS), water, 30% Acrylamide, 10% APS and TEMED. Once this is poured
and polymerized, 5% stacking gel containing 4x stacking buffer (0.25M tris pH6.8, 0.4%
SDS), water, 30% Acrylamide, 10% APS and TEMED was layered over the 12%
resolving gel and allowed to polymerize with a comb inserted for well creation. This
polymerized gel was placed in an electrophoresis tank. rGFP samples (G0, G3, GCE, E2,
E3, W3, W4) with water and Sample Loading Buffer (SLB; 6.25 mM tris pH 6.8, 4.5%
SDS, 0.05% v/v Beta-mercaptoethanol, 50% V/V glycerol, and 0.01% w/v bromophenol
blue) were prepared and loaded into the wells of the gel along with a molecular weight
ladder. Electrophoresis was performed at 200 volts for approximately 45 minutes.
Coomassie blue was then used to stain and de-stain the gels so that the protein bands
could be visualized. The molecular weight ladder allowed us to evaluate the relative
molecular weight of the visible proteins and the purity was estimated based on the
thickness of the bands seen.

Western Blot and Protein Verification:

Another SDS-PAGE gel was prepared and the samples were prepared by adding Betamercaptoethanol and loaded onto the gel. These protein bands were then transferred to
the nitrocellulose membrane, which was then stained and destained with Ponceau S dye
to visualize the bands. The membrane was blocked with 5% non-fat dry milk/TBS
solution and washed with 0.05% Tween 20/TBS several times. Then the membrane was
probed with mouse IgG anti-Xpress epitope Mab solution which was the primary
antibody and washed several times. The secondary membrane Sheep IgG anti-mouse
IgG conjugated horse radish peroxidase polyclonal anti-serum solution was incubated
with the membrane. This was then washed several times with 0.05% Tween 20/TBS and
finally washed with just TBS. The western blot was then developed with TMB substrate
solution and then the reaction was stopped with water and the results were recorded.

Results

The rGFP was expressed in E. coli by taking the G0 sample at time 0 before induction
and inducing expression with IPTG. The G0 sample has the Lac repressor, which inhibits
the T7 RNA polymerase therefore, preventing the GFP from being expressed. The G3
sample is collected 3 hours post-induction with IPTG. IPTG inhibits the lac repressor,
which allows the T7 RNA polymerase to bind the T7 promoter commencing transcription
to express His6-Xpress-GFPuv. The pRSETA-GFPuv plasmid also has an Ampicillin
resistant gene, which maintains selection of the rGFP plasmid and prevents
contamination. The T7 promoter, rGFP gene and Ampicillin resistance gene can all be
visualized on Figure 1 (see attached), which is a plasmid map of pRSETA-GFPuv.

The rGFP is diagrammed schematically in Figure 2 (see attached), the N-terminus at the
5 end is labeled followed by the His6 Xpress Epitope tag, which spans amino acids 5
through 39. The DNA sequence of GFPuv then follows with 238 amino acids including
the chromophore from amino acids 103-105 which is the cause of fluorescence.

Figure 3 (see attached) shows the combined elution profile of the relative fluorescence
and the total protein amount in the washes (W1-W6) and elutions (E1-E6) that were
purified through the Ni2+ agarose column with a bed volume of 0.5 mL. The protein
amount of rGFP was found by the Bradford assay and E2 had the highest fluorescence
with 129907.5 RFUs and W3 had the highest protein amount of 202 ug.

Figure 4- SDS-PAGE gel of

rGFP samples using the 12% or
rGFP fractions and Coomassie
Blue staining

Figure 4 and Figure 5 (see

attached) both show the SDSPAGE gel using 12% resolving
gel. Figure 5 is an enlarged scaled
version of Figure 4. The band
representing rGFP is highlighted in
Figure 5 and the relative molecular
weight based on the standard curve
was found to be approximately
37kD. The purity based on the gel
figures was estimated to be about
55% for E3 by measuring the
thickness of the bands in the E3 lane. This allowed for the calculation of rGFP yield and
was found to be 47.2ug.

Figure 6- Western Blot of Analysis of rGFP Fractions

The Western blot results in Figure 6 shows the fractions G3, GCE, E2, E3, W3 and W4 to
have strong bands from 35-37kDa, which confirms the presence of rGFP and rules out
the possibility of a contamination or degradation because the molecular weight or rGFP is
close to this value.

Conclusion

The estimated molecular weight of rGFP was calculated to be 33.5 kDa and in the
experiment, the relative molecular weight using SDS-PAGE was found to be 37 kDa so
relatively close to the estimated value. E2 and E3 gave the highest fluorescence of the all
the washes and elutions which was expected. The purity of rGFP in E3 was found to be
55%, which probably should have been higher. The highest total protein amounts were
found in the wash fractions, which was expected because the point of the washes was to
wash out all of the non-rGFP protein leaving most of the rGFP to come out in the

elutions. The western blot did confirm the presence of rGFP so the experiment did work
and we did not purify a contaminant or degraded version of rGFP.

The use of rGFP can be used in further research where it could be linked to any gene of
interest. An experiment to continue the understanding of GFP would be to try and modify
the chromophore so that GFP could fluoresce a range of colors and can then be used to
tag different processes simultaneously. GFP can also be used to track the location of cells
in different organisms based on its fluorescence properties, another experiment could be
done linking GFP to cancer cells in order to monitor growth and metastasis so that
treatments could be targeted to the specific areas of interest.

GFP will make a large impact in the field of science because exogenous substrates and
cofactors are not required for this fluorescence; GFP expression can be used to monitor
gene expression and protein localization in living organisms. (Chalfie, Tu and
Euskirchen) The fluorescence labeling via gene fusion would be site-specific and could
eliminate the present need to purify and label proteins in vitro and microinject them into
cells. (Heim, Prasher and Tsien)

Bibliography
Chalfie, Martin, et al. "Green Fluorescent Protein as a Marker for Gene Expression."
Science 263 (1993): 802-805.
Heim, Roger, Douglas C. Prasher and Roger Y. Tsien. "Wavelength mutations and
posttranslational autoxidation of green fluorescent protein." Proceedings of the National
Academy of Sciences 91 (1994): 12501-12504.
Prasher, Douglas C., et al. "Primary structure of the Aequorea victoria green-fluorescent
protein." Gene (1992): 229-233.
Rippel, Scott. "Biochemistry Lab Lecture Notes." Richardson: The University of Texas at
Dallas, 7th November 2014.