1. Crystallization of Chromatographically Purified 6X-His-tag Green
Fluorescent Protein via Induction of pET21b Plasmid from Transformed
BL21(λDE3) Escherichia coli.
Cameron Naglieri-Prescod
Biochemistry 276
05/03/2013

2. 2
Abstract
Green Fluorescent Protein is a very useful and interesting tool for scientists to express
genes and proteins, and visualize observations of those proteins. In this study, goals of the
experiment were to amass a substantial amount of 6X-His-tag GFP, and to crystallize the GFP.
The 6X-His-tag GFP gene in a BL21(λDE3) E.coli bacterial culture was induced to produce
GFP. Via cell lysis, nickel affinity chromatography and hydrophobic interaction
chromatography, the GFP was obtained in a purified form. Different solutions from the
chromatography methods were exposed to a Bradford Assay and a Sodium Dodecyl Sulfate-
Polyacrylamide Gel Electrophoresis (SDS-PAGE) to quantitatively determine the concentrations
and amounts of total protein in different solutions, and to determine if there is GFP present and
the size of the GFP in the solutions. A standard curve and best fit line were obtained via the
Bradford Assay. Hanging drop vapor diffusion crystallization of the purest GFP solution was
performed with a lysozyme crystal comparison, and the superlative crystals of both the lysozyme
and GFP achieved were photographed and observed.

3. 3
Introduction
In the world's oceans, there are numerous creatures that exhibit bright and fascinating
colors and patterns. These creatures use these colors and patterns for a multitude of tasks, from
finding a mate, to camouflage, to an indicator of danger. There is one creature that uses color in
a slightly different manner. The crystal jellyfish, also known as Aequorea victoria, is a
simplistic and delicate jellyfish that uses bioluminescence to glow and produce light. What
makes this bioluminescence possible is a small protein appropriately named Green Fluorescent
Protein, or GFP, because of the emission of green light this protein exerts at a certain
wavelength. GFP is used in the biochemical field as a fascinating indicator for the expression of
genes and also other proteins. Scientists now are creating perceivable three-dimensional
structures of proteins to identify the properties and functions of these proteins at a level smaller
than the human eye can see.
A set of laboratory experiments were conducted to extract green fluorescent protein from
Escherichia coli (E.coli) that has been genetically modified to produce abundant amounts of
whatever protein desired in a controlled setting. The main goals of the experiment were to amass
a substantial quantity of green fluorescent protein at a high concentration, and to ultimately
crystallize the extracted GFP.
The E.coli used for the experiment, named BL21(λDE3), was engineered to produce a
large amount of protein. The engineered plasmid used, named pET21b, contains a T7 viral
promoter in its sequence, and this promoter is transcribed by a T7 RNA polymerase; the
polymerase is specific to the T7 promoter and therefore will not transcribe any other sequence
but that promoter sequence. While the T7 promoter is influenced by the T7 RNA polymerase,
the polymerase is controlled by an inducible bacterial promoter, specifically for the lac operon
gene on pET21b. The inducer for the bacterial promoter is isopropylthiogalactopyranoside
(IPTG). This compound behaves much like lactose in terms of expression of genes, but it is not
metabolized so it remains intact. The T7 promoter on pET21b resides next to the multiple
cloning site, which in turn resides by the sequence for a specific GFP gene, 6X-His-tag GFP
(GFP with six histidine residues attached). The result of specificity in sequencing is a large
amount of mRNA, which leads to an immense amount of GFP translated. Induction of the GFP
gene was performed on a colony of BL21(λDE3) to express the GFP gene in the pET21b
plasmid. Cell lysis was performed on the sample in order to extract the induced 6X-His-tag GFP
from the plasmids to continue with the purification processes and crystallization; a cell pellet and
a supernatant (cleared lysate) were obtained via lysis.
Two different methods of purification were implemented to expel the contaminant
proteins and to extract a sample of pure GFP. The first method used was Nickel Affinity
Chromatography. This method uses a molecule called imidazole, which binds to the 6X-His-tag
GFP and keeps it from being washed out of solution. This was used to purify the GFP that was
contained in the supernatant that resulted from the cell lysis. Different concentrations of
imidazole allowed for higher purification, but lower concentration of GFP. To further purify and
to concentrate the sample, Hydrophobic Interaction Chromatography was utilized. This method
is based on the idea of salting out proteins. The step adds a high concentration of salt to make

4. 4
proteins insoluble, and these proteins are then discarded when the salt concentration is changed.
In this case, the salt concentration is set high enough so that contaminants are eliminated from
solution but the GFP remains in solution. Both the nickel affinity and the hydrophobic
interaction chromatography steps purify the specific target protein and concentrate it as well.
Qualitatively the chromatography steps show a highly concentrated GFP sample, but
quantitatively there is not enough information. Direct Ultraviolet spectrophotometry of protein
samples is a widely used method as an estimator for protein concentration, but it does not
pinpoint the exact amount. To obtain a direct numerical concentration, a Bradford Assay was
conducted. This procedure uses the binding properties of Coomassie Brilliant Blue reagent to
bind to protein in order to determine a concentration of total protein in a sample; Coomassie
binds to all of the proteins in the sample, so it is not possible to distinguish between the proteins.
The protein bovine serum albumin was used in this assay as the control for the standard curve.
This assay lacks the capability of distinction of individual proteins in a sample, but does provide
an average optical density (OD) of the sample. The information obtained is used to create a
standard curve and a linear range of the sample, and this curve allows for the comparison
between absorbance value and protein concentration.
In order to determine the purity of the GFP sample, a Sodium Dodecyl Sulfate-
Polyacrylamide Gel Electrophoresis (SDS-PAGE) was conducted. The basic concept of
electrophoresis is the progression of charged particles exposed to an electric field. The SDS-
PAGE tracks the mobility of the target protein by denaturing the protein into a primary structure.
The negatively charged sodium dodecyl sulfate disrupts the tertiary and quaternary structures of
proteins, and when the protein is exposed to a positive charge, the protein has an overall
negative charge due to the SDS. When the protein is inserted into the gel, and when a positive
electric field is passing through, the protein will drift towards the positive terminus at a rate that
corresponds to their molecular weight; a blue dye is inserted to track the protein migration. The
result of the SDS-PAGE procedure is distinct bands of protein based on molecular weight.
The experiment concludes with the crystallization of the 6X-His-tag GFP. Crystallization
requires the protein to have a high concentration to ensure protein growth, but also the right
amount of a precipitant to lower the overall solubility of the protein. One of the most common
methods of crystallization is the hanging drop vapor diffusion method, which utilizes
evaporation. The protein is mixed with a precipitant in a certain ratio, sealed in a container, and
left alone to crystallize. Through vaporization and condensation of the precipitant, the
crystalline structure of the protein grows while the concentration of the protein increases. The
end result is an array of different protein crystals for observation.
Based on the various types of purification and testing for protein concentration, a large
quantity of 6X-His-tag GFP in a very high concentration is expected to be acquired as a product
of the entire procedure, and a large amount of high-concentrated crystallized GFP is expected to
result as well from the crystallization method used in the experiment.

5. 5
Materials and Methods
Bacterial Strain and Reagents
The bacterial strain used for the experiment was a pET system host strain called
Escherichia coli BL21 (λDE3). The genotype of Bl21(λDE3) is F-ompT hsdSB (rB- mB-) gal
dcm (λDE3). The encoding of the T7 RNA polymerase gene and the integration of the λDE3
lysogen which also contained T7 RNA polymerase gene under the influence of a lactose-
inducible lacUV5 promoter on this specific BL21 strain, allowed for the high expression of a
target protein, in this case Green Fluorescent Protein (GFP), in an a posteriori manner. The
plasmids used for this experiment were pET plasmids that contained the GFP gene, which
encoded for the production of Green Fluorescent Protein. The plasmid that contained the GFP
gene, called pGFPstop, was a gift from Professor Jeanne Hardy, Department of Chemistry,
University of Massachusetts. The GFP gene was subcloned into the multiple cloning site
pET21b, which was obtained from Novagen located in Darmstadt, Germany, the coding region
of GFP was presented in the most accurate reading frame, and GFP was fused with a 6X His tag
located at the C-terminus of that GFP. All of the reagents and supplies needed for this
experiment were acquired from Fisher Scientific (Fair Lawn, New Jersey), unless stated
otherwise.
Induction of GFP expression
A group of BL21(λDE3) that was transformed with pETHis GFP was used by the
teaching staff to inoculate a 5 mL culture of LB medium that contained 100 μg/mL ampicillin
antibiotic (5 μL of a 100 mg/mL stock in 5 mL of medium). The culture was then shaken at 200
rpm in an overnight period at 37ºC to promote growth of the culture.
2 mL of the overnight culture was diluted the next day in 500 mL of fresh LB + antibiotic
at room temperature. The culture was then incubated with a shaking period of approximately 3
hours at 200 rpm at 37ºC to ensure an optical density (OD595 ) of 0.5-0.7. The inducer of the
bacterial promoter that controlled the T7 promoter was IPTG (isopropylthiogalactopyranoside).
This inducer was added to a final concentration of 0.1 mM, and the cultures were shaken at 200
rpm at room temperature for a second night (12-16 hours).
After the 12-16 hour period, 500 mL centrifuge containers were pre-weighed, and the
culture was poured into the containers to the neck of the container. The containers were
balanced to within 0.1 mL with the cap on the container. The cells were collected via
centrifugation at 5,000 rpm for 15 minutes at 4ºC. The clear culture medium that resulted from
centrifugation was decanted and removed. The remaining culture was equally distributed into
centrifuge bottles, balanced, and harvested via centrifugation at 5,000 rpm for 15 minutes at 4ºC.
The clear culture medium was decanted and removed, and the weight of the pellet at the bottom
of the centrifuge bottle and the weight of the centrifuge bottle was recorded.

6. 6
The pellets were then frozen by placing the bottle in a freezer at -80ºC for several hours.
The pellet was removed by tipping the bottle in lukewarm tap water and "popping" the pellet
with a spatula into a 50 mL conical tube and placed back into a -80ºC freezer. The tube was
labeled with the weight of the pellet. All of the steps above were performed by the Introductory
Biochemistry Laboratory teaching staff at the University of Massachusetts, Amherst.
Cell Lysis
A frozen pellet was obtained from a teaching assistant, and was thawed to room
temperature for 15 minutes. The mass of the cell pellet was 2.50 grams. The cell pellet was re-
suspended in room temperature BugBuster reagent via gentle pipetting using 5 mL of BugBuster
per gram of cell pellet (BugBuster HT with Benzonase nuclease was purchased from Pierce in
Darmstadt, Germany). With 2.50 grams of the cell pellet multiplied by 5 mL of BugBuster
reagent per gram of cell pellet, a volume of 12.5 mL of BugBuster was added to re-suspend the
cell pellet. 1.25 mL of a 10 mg/mL lysozyme solution was added so that the final concentration
was a ten-fold solution at 1 mg/mL. Lysozyme was purchased from Sigma-Aldrich in St. Louis,
Missouri. Digestion of the cells then went for 15 minutes at room temperature.
The tube was then balanced within 0.1 g with a conical tube filled with water, and was
then centrifuged for 30 minutes at 15,000 x g, or about 9000 rpm, at 4ºC. The supernatant was
collected and transferred into a new tube. Only half of the green colored supernatant was used
for the experiment; the other half was used with another group conducting the same procedure.
500 μL of the cell lysate (supernatant) was saved in a microfuge tube for further
experimentation, and was stored at -20ºC. The remainder of the supernatant was used for
purification.
Nickel Affinity Chromatography
A 10 mL syringe was filled with Buffer NPI-10
(50 mM NaH2PO4, 300 mM NaCl,10 mM imidazole, pH 8.0). A suitable adapter was attached
to the syringe, and air was expelled by depressing the plunger of the syringe until Buffer NPI-10
dripped from the tip of the adapter. The syringe was then attached to a Ni-NTA Superflow
cartridge inlet (Qiagen, Valencia, California), and the outlet stopper of the cartridge was
removed. The cartridge was then equilibrated with 10 column volumes, or 10 mL, of Buffer
NPI-10, at a flow rate of 1 mL/min. The liquid passed through the syringe was placed in a test
tube marked "Waste", and was discarded.
The syringe was removed and filled with cleared lysate solution at the same flow rate of 1
mL/min. This liquid was collected in a test tube labeled "AFT", or affinity flow-through
fraction. 1 mL of the fraction was transferred into a microfuge tube and was labeled "AFT".
The tube was stored at 4ºC for future use.
The cartridge was washed with 10 column volumes (10 mL) of Buffer NPI-20
(50mM NaH2PO4, 300 mM NaCl, 20 mM imidazole,pH 8.0) using a fresh syringe and applying

7. 7
the same flow rate of 1 mL/min as both the waste equilibration and the affinity flow-through.
The liquid was collected into a test tube labeled "Wash", which represented the affinity wash
fraction. 1 mL of the fraction was transferred into a microfuge tube and was labeled "AW". The
tube was stored at 4ºC for future use.
A syringe was filled with ~7 mL of Buffer NPI-250
(50mM NaH2PO4, 300 mM NaCl, 250 mM imidazole,pH 8.0), and elute protein from a
cartridge at a flow rate of 1 mL/min. 1 mL was collected at a time and separated into individual
test tubes labeled "AE 1", "AE 2" etc...(approximately 5-7 tubes). "AE" stands for affinity
eluate. The collection ceased as the green color of the eluate began to diminish. The highest
concentration of green eluate was combined into a 15 mL conical tube, and was mixed gently.
The 500 μL of the combined eluate was stored at 4ºC for future use. The remainder of the
combined eluate was used for a hydrophobic interaction column. The final volume of the
combined affinity eluate was 2.4 mL. The fractions collected through Nickel Affinity
Chromatography were 1.0 mL of the affinity flow-through (AFT), 1.0 mL of the affinity wash
(AW), and 0.5 mL of the affinity eluate (AE).
Hydrophobic Interaction Chromatography
The remainder of the affinity eluate was brought to 2M (NH4)2 SO4 by adding an equal
volume of a 4.1mM saturated solution of (NH4)2 SO4. The stoppers in the column were removed
and were placed in a safe place to avoid losing the small pieces. A HiTrap ButylS-FF 1 mL
sized cartridge was attached to a syringe, which was filled with 5 column volumes of RODI H2O,
and was run through the column in order to remove any solution that was previously in the
column (HiTrap ButylS-FF 1 mL cartridge was purchased from Amersham/GE Healthcare in
Piscataway, New Jersey). The flow-through was then discarded. The syringe was then loaded
with 5 column volumes of 2M (NH4)2 SO4. The flow-through was then discarded.
A fresh syringe was used to acquire 10 column volumes of 2M (NH4)2 SO4. This
solution was run through the column, and the flow-through was collected in a test tube labeled
"HI Wash" which stands for "hydrophobic interaction wash". 1.0 mL of the HI Wash solution
was transferred into a microfuge tube and was stored at 4oC for future experimentation.
A fresh syringe was used to acquire the pooled GFP affinity eluates onto the column.
The flow-through was then collected and set aside in case any error occurred. A clean syringe
was loaded with 5 mL of RODI H2O, and was run through the column. The liquid was collected
in microfuge tubes labeled "HIE 1", "HIE 2" etc... "HIE" stands for hydrophobic interaction
eluate. The microfuge tubes were switched as the concentration of green color changed; this was
to acquire the highest concentration of protein. 100 μL of the highest concentrated GFP solution
was then transferred into a microfuge tube, labeled, and stored at 4oC for future use. The
remaining HI eluate, which was pure GFP, was also stored at 4oC in a separate tube. A final
excess volume of 500 μL was recorded. The fractions collected through Hydrophobic
Interaction Chromatography were 1.0 mL of the hydrophobic interaction wash (HIW), 100 μL of
the hydrophobic interaction eluate (HIE), and the remainder of the concentrated HI Eluate.

8. 8
Bradford Assay
Bradford reagent used for the assay was purchased through Biorad in Hercules,
California. Bovine serum albumin (BSA) was obtained from Sigma-Aldrich in St. Louis,
Missouri. All other materials were acquired from Fisher Scientific in Fair Lawn, New Jersey.
A 96 well microplate was obtained and was oriented with well A1 on the top left corner.
50 μL of RODI H2O was inserted into wells A1 and A2 for reference and blank subtraction for
the wells. 100 μL of 1 mg/mL bovine serum albumin (BSA) was inserted into B1 and B2. 100
μL of each of the samples made from both the Nickel Affinity Chromatography and the
Hydrophobic Interaction Chromatography were added into the wells B3-B8 in the following
order: B3 had cleared lysate, B4 had Affinity Flow-through, B5 had Affinity Wash, B6 had
Affinity Eluate, B7 had HI Wash, and B8 had HI Eluate (B8 was 50 μL of eluate and 50 μL of
H2O, because there was only 100 μL of concentrated HI Eluate to begin with).
Approximately 20 mL of RODI H2O was poured into a tray for the multichannel pipettor.
The multichannel was set to pipet mode and the volume set was 50 μL; 50 μL of RODI H2O was
inserted via multichannel pipet into all of the wells in rows C-H. 50 μL of the solutions in row B
were removed and added to row C. The solution was mixed and 50 μL of the solutions in row C
were removed and added to row D. This procedure was repeated until 50 μL of row G was
transferred into row H. 50 μL of the solutions in row H were removed and discarded, leaving all
of the sample wells with a volume of 50 μL. 110 μL of RODI H2O was added to every well to
bring the total volume of each well up to 160 μL.
Approximately 5mL of Bradford reagent was inserted into a plastic reservoir, and 40 μL
of the reagent was placed into each well starting from row H up to row B (cautionary
measurements were used to not stain any of the equipment or anyone using or not using the
Bradford reagent). Bubbles that were created by the addition of reagent were removed by
pulling up 5 μL ethanol (C2H5OH), and popping the bubbles on the surface, making sure not to
insert any ethanol into the wells. The microplate was put into a plate reader, and the optical
density (OD) values of the wells were measured. A graph of the BSA standard curve was
generated with the average OD values, and the equation y = mx + b was used to describe the line
which resulted from the information. The equation was also used to determine the concentration
of protein in each of the fractions. The plot of the graph made was μg of BSA on the x-axis
versus average OD values on the y-axis. The concentrations of the fractions were determined
and the remainder of the fractions was stored in a 4oC environment.
Sodium Dodecyl Sulfate- Polyacrylamide Gel Electrophoresis (SDS-PAGE)
Calculations were made during the Bradford Assay period to determine how much of
each fraction was needed to load on the Polyacrylamide gel. 4 μL of 5X SDS Sample Buffer
were added to 7 different microfuge tubes. The amount of fraction and amount of water added
for each specific tube is as follows: 5 μL of Protein Marker and 11 μL of water for tube 1, 2.1 μL
of cleared lysate and 13.9 μL of water for tube 2, 2.4 μL of AFT and 13.6 μL of water for tube 3,

9. 9
13.4 μL of AW and 2.6 μL of water for tube 4, 13.8 μL of AE and 2.2 μL of water for tube 5,
16.0 μL of HIW and 0.0 μL of water for tube 6, and 6.9 μL of HIE and 9.1 μL of water for tube
7. The samples were heated at 100oC for 5 minutes for complete denaturing of the proteins. The
samples were then placed immediately on ice until it was necessary to load the gel. The tubes
were spun in a microfuge for ~5 seconds to collect all of the liquid to the bottom of the tubes.
For the Polyacrylamide gel set up, the center chamber was filled with electrophoresis
buffer (1X SDS Gel Buffer), and proper gel fit and orientation was made by the teaching
assistants. The gels used were 12% Tris-HCl ready gels; no gels were poured The 1X SDS Gel
Buffer was 25 mM Trizma base, 192 mM glycine, and 0.1% sodium dodecyl sulfate (SDS). The
5X SDS Sample Buffer was 250 mM Trizma base, 500 mM glycine, 10% sodium dodecyl
sulfate (SDS), 0.5% bromophenol blue, and 50% glycerol. The 12% Tris-HCl ready gels were
purchased from Biorad in Hercules, California. The EZ-Run prestained protein molecular
weight markers were from Fisher Scientific in Fair Lawn, New Jersey. The wells of the gel were
loaded left to right (the well numbers corresponded with the tube numbers), starting with tube 1
containing the Protein marker solution, tube 2 containing the cleared lysate, tube 3 which had the
Affinity Flow-through, tube 4 which contained the Affinity Wash, tube 5 which contained the
Affinity eluate, tube 6 which had the HI Wash, and tube 7 which contained the HI Eluate
solution. Wells 8 and 9 were loaded with 1X Sample Buffer. The gels were run at 150 V or
approximately 45 amps, for around 35 minutes, or until the blue loading dye was near the
bottom. The gels were removed from the casing and buffer with the aid of teaching staff
(extreme caution was used when handling the gel, not only because it is fragile, but also because
unpolymerized acrylamide is a potent neurotoxin; extreme caution and safety is advised).
The gels were transferred into a plastic container, which were then inserted into a
microwave with added RODI H2O, and was set for ~90 seconds or until boiling commenced.
The water was removed, more water was added, and the procedure of boiling was repeated. The
water was removed, fresh water was added, and was left on a rotator platform for 5 minutes. The
water was poured off, and GelCode Blue was added to cover the gel. The gel with the dyeing
solution was put in the microwave until it boiled. The container was then placed back on the
rotator platform until it cooled. After cooling, the stain was removed and fresh water was added.
A picture was taken of the finished product.
Further Concentration of GFP
Gel images from the SDS-PAGE and the appearance of GFP in the HIC eluate were used
to determine which of the GFP samples eluted from the hydrophobic interaction columns
contained the most concentrated GFP. The GFP samples created in the lab were pooled, and ~10
mL of GFP was dialyzed in 2 L of 10 mM Tris, pH 8.0 for an overnight period at 4oC. The GFP
was then concentrated to a 10 mg/mL concentration in a 7 mL Pierce protein centrifugal
concentrator via centrifugation. The protein centrifugal concentrator was set at a speed of 5000 x
g for 35 minutes at 4oC in a swinging bucket centrifuge.

10. 10
Protein Crystallization
For both the crystallization of the lysozyme and GFP, the hanging drop vapor diffusion
method was used to optimize the amount of crystallization of protein. The initial concentration
of lysozyme was 1 µL of 50 mg/mL protein solution in 10 mM sodium acetate buffer, pH 4.6.
The initial concentration of GFP was 1 µL of 10 mg/mL GFP in water ((NH4)2SO4 traces were
present due to the previous purification process). The final concentrations of the lysozyme
reagents for lysozyme crystal formation were 0.25 M NaCl, 100 mM sodium acetate, pH 4.6, and
2 M NaCl, 100 mM sodium acetate, pH 4.6. The final concentrations of the GFP reagents for
GFP crystal formation were 10% (weight/volume) polyethyleneglycol (PEG) 8000, 50 mM
MgCl2, 100 mM TrisCl pH 8.2, and 40% (weight/volume) polyethyleneglycol (PEG) 8000, 50
mM MgCl2, 100 mM TrisCl pH 8.2. Both the lysozyme and GFP crystals were grown using the
hanging drop vapor diffusion method. A glass coverslip sat on top of a well filled with
precipitate. On the coverslip underneath, 1µL of protein was mixed with 1 µL of the specific
precipitant, and the lid was flipped and sealed with VDX grease.(VDX greased crystallization
plates and siliconized circular coverslips were bought from Hampton Research in Aliso Viejo,
California. The ratios of protein to precipitant were altered from (1 protein: 1 precipitant), (2
protein: 1 precipitant), and (1 protein: 2 precipitant) for all 24 wells, in turn creating 72 possible
outcomes. After the crystallization wells were set up, the protein spots were observed under a
microscope to detect any early crystal formation or any aggregate formation. The wells were
then stored in a cupboard at room temperature for a 1-week incubation period. After one week
the wells were removed from the covered to observe both the lysozyme crystals and the GFP
crystals. The best samples were determined and photographs of the most successful crystals
were taken.
Results and Discussion
A large culture of lac-regulated construct encoding 6X-His-tag GFP was exposed to
isopropylthiogalactopyranoside (IPTG) in order to produce a high level of expression of the
GFP. In order to extract the GFP, a cellular lysis of the pET21b plasmid located inside the
BL21(λDE3) E.coli was performed. The result of the cell lysis was a pellet of cellular debris and
a supernatant, or cleared lysate, containing the total amount of protein that was in the cells.
The cleared lysate produced from cell lysis was then exposed to first nickel affinity
chromatography, and then hydrophobic interaction chromatography. In the nickel affinity step,
three different samples were produced to test concentration of GFP; an affinity flow-through
(AFT), which had the protein that did not adhere to the nickel affinity column, an affinity wash
(AW), which washed away any contaminant protein, and an affinity eluate (AE), which
contained the GFP from the chromatography; this eluate glowed green. For the hydrophobic
interaction chromatography, two samples were produced, the hydrophobic interaction wash
(HIW), which removed more contamination, and the hydrophobic interaction eluate (HIE),
which housed the purified and concentrated GFP; this eluate glowed an intense neon green color.
In total, the nickel affinity chromatography and the hydrophobic interaction chromatography
resulted in six samples; a cleared eluate with all protein present, an affinity flow-through with

11. 11
little GFP, an affinity wash with very little GFP, an affinity eluate with a substantial amount of
GFP but not concentrated, a hydrophobic interaction wash, which contained very little traces of
GFP, and a hydrophobic interaction eluate, which had a large amount of GFP in a very high
concentration.
A Bradford Assay was conducted to quantitatively measure the total protein
concentration in each of the samples made via both chromatography steps and the cleared lysate.
Bovine serum albumin (BSA) was the protein used to create the standard curve for the Bradford
assay, and the Coomassie dye used for the assay binds non-specifically to all the protein in the
sample, so there is no determination what is GFP and what is not. The result of the assay is
shown below.
In the Bradford assay standard curve above (Fig.1), as the concentration of bovine serum
albumin increases, the absorbance value also increases. This correlation allows for the
determination of exactly how much protein is needed to run a polyacrylamide gel
electrophoresis. The straight line in the graph is the best fit line of the data. The equation of the
line is y=0.0062x+0.0238. An R2 correlation coefficient was calculated to express the linear
regression of the observed data points. Because the value is 0.9015, the best fit line exhibits a
strong, positive, and linear correlation to the data points. With the equation obtained from the
Bradford assay, the amount of protein of each individual dilution was calculated in order to
determine how much protein was needed for the polyacrylamide gel electrophoresis. The table
below shows the average concentration of protein in each dilution, and the amount of protein in
μg needed for the SDS-PAGE.

12. 12
* There was very little protein concentration in the HIW to acquire the optimal value of ~40μg.
In Table I above, the top row of numbers on the table shows the average protein
concentration calculated for each of the samples. The middle row divides the average
concentration by 50 to remove the water from the equation, and to have a more concentrated
number for easier calculations. The bottom row of the table shows the amount of protein loaded
for each sample, which in almost all of the samples, was close to the target protein amount,
which was 40 μg. The method of determining the amount of protein is to multiply the protein
concentration by the volume needed to be loaded into the gel. The only amount of protein that
differed from the expected amount was the protein in the hydrophobic interaction wash; there
was very little protein and a lot of volume, and the maximum capacity of a well in SDS-PAGE is
16 μL, so 4.7 μg was obtained by using 16 μL for the volume necessary for the loading of the
gel.
A Sodium Dodecyl Sulfate- Polyacrylamide Gel Electrophoresis was performed to
determine the purity of the 6X-his-tag green fluorescent protein in each of the samples. The
apparatus is comprised of a container with two electrodes, a negative on the top and a positive on
the bottom, a casing for the gel, and the polyacrylamide gel, which can be made or bought; a pre-
made gel was used to save time. Seven wells were loaded with 16μL of a solution of buffer,
protein and water. The results of the SDS-PAGE is shown below in Figure 2.
Table I: Chart of average protein concentration via Bradford Assay and amount of protein loaded
for each Polyacrylamide Gel sample.
Cleared
Lysate
Affinity
Flow-
Through
Affinity
Wash
Affinity
Eluate
Hydrophobic
Interaction
Wash
Hydrophobic
Interaction
Eluate
Average
Protein
concentration
(μg/μL)
966.7097 829.9355 148.7419 144.6866 14.83871 287.8065
Average
Protein
concentration /
50
19.33419 16.59871 2.974839 2.893733 0.296774 5.756129
Amount of
protein (μg)
loaded for each
sample
40.6μg 38.4μg 39.8μg 39.9 μg 4.7 μg* 39.7μg

13. 13
The leftmost lane in the figure represents the protein marker as an indicator for where the
protein will be interpreted. Lane 2 contains the cleared lysate solution created from the cell lysis
of the BL21(λDE3) E.coli culture. The figure shows that there are multiple proteins of multiple
sizes, as there is a single blue streak going down the column. Lane 3 contains the affinity
chromatography flow-through solution. The figure shows that there are multiple proteins of
multiple sizes, as there is a blue streak going down the column. There is a high concentration of
protein located in the 36-38 kDa (kiloDaltons) range, but it is not GFP, as GFP stops at the 26-34
kDa range. Lane 4 contains the affinity chromatography wash solution. The figure shows that
there are multiple proteins of multiple sizes, as a single blue streak descends down the column.
Overall the concentration is much less than the previous wells, as it is a wash and it is not
supposed to contain much protein. Lane 5 contains affinity chromatography eluate solution. The
figure shows that there is a high concentration of protein in the 26-34 kDa range, indicating that
there is an abundance of GFP present. Lane 6 contains the hydrophobic interaction
chromatography wash solution. The figure shows that there is little to no protein present
indicating that the purity of the protein is very good. The last lane contains the hydrophobic
interaction chromatography eluate solution. The figure shows that there is a high concentration
of protein in the 26-34 kDa range, indicating that there is an abundance of GFP present. This

14. 14
particular eluate is known to be very pure based on the hydrophobic interaction affinity wash
lane, which contained little to no protein in the column.
A protein crystallization procedure was performed in order to grow GFP crystals and
observe the structural patterns of crystallized GFP. Two different proteins were used for
crystallization; the main purpose being to compare structures of two different proteins. The
proteins used were purified 6X-His-tag GFP, and the enzyme lysozyme. A set up of 24
individual wells was used. The method used was the hanging drop vapor diffusion method,
which is a sample of protein and precipitant hanging on a cover slip, which is sealed and left
alone to crystallize. Seven of the wells were for lysozyme, and the other 17 wells were for GFP.
Table II below shows the schematic of the wells, what concentrations of precipitants were used
and where, and the protein to precipitant drop ratio for the crystallization.
Table 2: Table ofvolumes ofprecipitants in crystallization wells, and the concentration ratios of
protein to precipitant.
1* 0μL A
500 μL B
1:1
1:2 2:1
2 100 μL A
400 μL B
1:1
1:2 2:1
3 200 μL A
300 μL B
1:1
1:2 2:1
4 300 μL A
200 μL B
1:1
1:2 2:1
5 400 μL A
100 μL B
1:1
1:2 2:1
6 500 μL A
0 μL B
1:1
1:2 2:1
7 250 μL A
250 μL B
1:1
1:2 2:1
8** 0 μL C
500 μL D
1:1
1:2 2:1
9 75 μL C
425 μL D
1:1
1:2 2:1
10 100 μL C
400 μL D
1:1
1:2 2:1
11 125 μL C
375 μL D
1:1
1:2 2:1
12 150 μL C
350 μL D
1:1
1:2 2:1
13 175 μL C
325 μL D
1:1
1:2 2:1
14 200 μL C
300 μL D
1:1
1:2 2:1
15 225 μL C
275 μL D
1:1
1:2 2:1
16 250 μL C
250 μL D
1:1
1:2 2:1
17 275 μL C
225 μL D
1:1
1:2 2:1
18 300 μL C
200 μL D
1:1
1:2 2:1
19 325 μL C
175 μL D
1:1
1:2 2:1
20 350 μL C
150 μL D
1:1
1:2 2:1
21 375 μL C
125 μL D
1:1
1:2 2:1
22 400 μL C
100 μL D
1:1
1:2 2:1
23 425 μL C
75 μL D
1:1
1:2 2:1
24 500 μL C
0 μL D
1:1
1:2 2:1
* Wells 1-7 contain lysozyme , solution A (0.25 M NaCl, 100 mM sodium acetate,pH 4.6) and solution B
(2.0 M NaCl, 100 mM sodium acetate,pH 4.6). The ratios refer to μL of protein per μL of A+B
precipitant in each well, resulting in three drops per well.
** Wells 8-24 contain GFP, solution C (10% (weight/volume) polyethyleneglycol (PEG) 8000, 50 mM
MgCl2, 100 mM TrisCl pH 8.2 and solution D (40% (weight/volume) polyethyleneglycol (PEG) 8000, 50
mM MgCl2, 100 mM TrisCl pH 8.2). The ratios refer to μL of protein per μL of C+D precipitant in each
well, resulting in three drops per well.
The asterisks below the table indicate the simplistic conditions of each well, such as the
type of precipitants used, what protein was used, and the drop ratios. The number on the left of
the well represents the well number. The two numbers on the right signify how much of each
solution was put into the well. The ratios on the bottom represent the three drops on the cover
slip, and the ratio of protein to precipitant. Wells 1-7 observed crystalline growth of lysozyme.
The only factor that changed per well was the amount of A + B precipitants in each well.
Precipitant A was 0.25 M NaCl, 100 mM sodium acetate, pH 4.6 and precipitant B was 2.0 M
NaCl, 100 mM sodium acetate, pH 4.6. A+B was only used with lysozyme. In wells 8-24, there
was observed crystalline growth of GFP. The only factor that changed per well was the amount
of C+D precipitants in each well. Precipitant C was 10% (weight/volume) polyethyleneglycol

15. 15
(PEG) 8000, 50 mM MgCl2, 100 mM TrisCl pH 8.2, and precipitant D was (40%
(weight/volume) polyethyleneglycol (PEG) 8000, 50 mM MgCl2, 100 mM TrisCl pH 8.2 C+D
was only used with GFP. The figure below shows the best result of the crystallization of both
the lysozyme and the GFP.
In the figure above, two different protein crystals were photographed to observe
noticeable differences. In Panel A, the shape of the lysozyme crystals are large cube shaped
crystals, are clear and are very defined. This specific well is comprised of 300μL of precipitant
A, and 200 μL of precipitant B. The drop ratio for this specific drop is a 2 μL drop of lysozyme
to a1 μL drop of A+B precipitant. In panel B, the shape of the GFP crystals are smaller, but are
bright green-yellow, and have a dual end fan shape to them. This specific well is comprised of
250μL of precipitant C, and 250μL of precipitant D. The drop ratio for this specific drop is a 2
μL drop of GFP to a 1μL drop of C+D precipitant.
The goals and aims of the protein purification and crystallization laboratory module were
to amass a substantial quantity of green fluorescent protein at a high concentration, and to
ultimately crystallize the extracted GFP. Through cell lysis, protein was obtained, and through
nickel affinity chromatography and hydrophobic interaction chromatography, the target protein
6X-His-tag GFP was purified and concentrated . The Bradford assay allowed for the
quantification of the protein obtained via lysis and chromatography. The SDS-PAGE helped to
determine the molecular weight of GFP, and to determine whether or not the protein in the
samples were indeed GFP. The hanging drop method for crystallization allowed for rapid
evaporation of the hanging drops, and increased concentration of crystallized lysozyme and GFP,
and produced spectacular crystals. Further testing of the GFP crystals to identify its structure
would be X-ray crystallography, which uses X-rays to create a stationary image of the billions of

16. 16
copies of the individual proteins. The crystallography also locates the electrons of the atoms that
make up the protein. The only disadvantage is that it does not detect moving parts. For that
nuclear magnetic resonance spectroscopy (NMR) is used, as it detects the moving parts of
proteins and gives the distances between atoms. With those two tools an accurate representation
of the desired protein is possible via creation of a three-dimensional structure. Scientists are able
to use the protein structures to look at diseases and diagnose treatment. That in turn allows for
the observation of the expression interaction between cells and proteins, specifically genes in the
cells, all thanks to a little green fluorescent protein found in a simple jellyfish. If this experiment
was done differently the amount of time for crystallization of the proteins would be longer to
ensure optimal crystal growth, and also to try different derivatives of GFP to see if there are
structural and chemical similarities. This experiment demonstrates that the methods of purifying
and crystallizing proteins are a very important asset in the biochemical field, as they help to
determine biological diagnoses and treatments at a molecular standpoint; this experiment showed
that proteins are fascinating molecules that majorly influence the way biological organisms work
and function, and that there are innumerable amounts of different types, all vital to the survival
of life.