The document describes the process of purifying the elongation factor LepA/EF4 protein from E. coli. The gene for EF4 was transformed into E. coli cells using a plasmid. The cells were then lysed using sonication and the EF4 protein was purified from the cell lysate using affinity chromatography and its hexahistidine tag. The concentration of the purified EF4 protein was determined to be 0.57 μg/μL using a Bradford assay and its molecular weight was found to be ~69 kDa by SDS-PAGE. Secondary and tertiary structural analysis using circular dichroism and fluorescence spectroscopy yielded thermodynamic values for EF4 protein denaturation.

1. 1
Protein Preparation of Translation Factors from E. coli
Eric R. Newman, Connor Stewart
Laboratory of Biochemistry, Bellingham Washington, 98225
November 24, 2014
Abstract
The gene coding for the elongation factor LepA/EF4 protein was
transformed into E. coli and over expressed using Isopropyl β-1-
thiogalactopyranoside (IPTG). After over-expression of the EF4 protein the E.
coli cells were lysed through sonication and separated by centrifugation.
Purification of the EF4 protein was done through the exploitation of the
hexahistdine tag engineered onto the expressed protein through the
transformation vector. The histidine tag was added to allow EF4 to be purified
from the cell lysate by affinity chromatography using a Nickel-Nitrilotriacetic
acid, Histidine binding resin (NI-NTA His-Bind resin). Concentration of the
affinity chromatography lysate was assessed through a BSA (Bovine Serum
Albumin) Bradford assay. The concentration was determined to be 0.57 µg/µL
and the molecular weight was determined by SDS-PAGE (sodium dodecyl
sulfate polyacrylamide gel electrophoresis) to be ~69 kDa. Also, a ~40 kDa
contaminate was identified in the purified lysate. Last, secondary and tertiary
structure thermodynamics of denaturation were assessed through circular
dichroism (CD) and fluorescence spectroscopy. Native and denatured samples
were analyzed with denaturation achieved through temperature for CD and
variation in urea concentration for fluorescence. Analysis yielded values of ΔGo'

2. 2
= 16 kJ/mol for the denaturation on the tertiary structure and ΔGo’ of 61 kJ/mol
for the denaturation of the secondary structure.
Introduction
E. coli is a gram negative rod shaped bacteria most widely known for the gastrointestinal
distress ingestion of certain strains of this bacterium induces. However, this bacteria is model
organism due to its quick lifecycle, ease of care, cheap cost, and relative simplicity in obtaining.
E. coli can be sustained on a variety of substrates. I is a facultative anaerobe which allows it to
survive both with and withouto oxygen. It also has an optimal growth temperature easily reached
by incubators. Most importantly is E. coli’s ability to transfer DNA through multiple pathways
(conjugation, transduction, and transformation) which allows the purposeful introduction of new
genetic material into the strain (Cao, et. al., 2014).
Plasmids are small circular sections of DNA mainly found within bacteria cells and can
replicate independently or be replicated within the bacterial DNA. Plasmids often contain
information to express new traits that increase survivability in various environments. The most
commonly known and exploited is antibiotic resistance. Plasmids can be transferred between
bacteria in multiple ways, such as cell conjugation, direct bridging between two bacterial cells.
Transformation a process of inserting genetic material into a cell from the extracellular
environment is another pathway of plasmid transference. This process is performed by shocking
the bacterium cell causing it to open its cell wall. Heat shock, incubating at a high temperature
and subsequently transferring the bacteria to a low temperature is an example of this method.
This is done in the hopes that the plasmid will make its way into the bacterium cells which can
then be selected through their antibiotic resistance as a selection trait (Cao 2014).

3. 3
Research on proteins has obstacles to overcome. Proteins often reside inside cells behind
the cell membrane and/or wall with many other macro/micro molecules. As such, lysing of cells
is a common method that releases the contents of the cytosol. This can be done with multiple
techniques depending on what type of cell is to be lysed. One such technique is sonication, the
use of high frequency sound waves to rupture the cell membrane and release cytosolic
components. High frequency sound waves give the lipid particles within the membrane bilayer
kinetic energy causing them to collide and rupture the cell membrane (Finer, 1972).
Additionally, the structure of proteins allows the selection of a desired protein from the
resulting cell lysate based on known properties. These properties include molecular mass, pI, or
structure of the protein. Using transformed cells and engineered plasmid, a protein with a desired
primary structural trait such as a hexahistidine, or GST tag can be achieved (Matzke, 1981). The
chemical properties of this tag can then be exploited to purify the target protein through affinity
chromatography (Scheich, 2003).
Polyacrylamide gel electrophoresis (PAGE) is a technique employed to determine the
purity of a sample, often proteins. The PAGE technique can also be applied to determine the
molecular weights of the compunds/proteins contained within the sample by comparing against
known standards (Weber, Osborn, 1969). Polyacrylamide gels separate the components of a
sample based on the size, weight and charge of the samples components. The acrylamide
monomer is induced to polymerize through the addition of an initiator-catalyst of ammonium
persulfate-N,N,N',N' and-tetramethyl-ethylenediamine (APS-TEMED). The size range of a gel is
dependent on its resultant pore size, which is directly linked to the concentration of acrylamide
within the pre-polymerized gel mixture (Candau, et. al. 1985).

4. 4
Sodium dodecyl sulfate (SDS) is added to the PAGE technique to denature protein
samples and mask their native charge by disrupting the non-covalent bonds of the native protein.
The SDS solvates the denatured protein with its negative sulfate group facing out giving a
uniform negative charge to the denatured protein (Reynolds, Tanford, 1970). This produces a
constant charge:mass ratio and a rod shaped protein-SDS complex, leaving sample mobility
through the polyacrylamide gel almost solely due to size of the sample as opposed to native
charge and tertiary/quaternary structure (Reynolds, Tanford, 1970).
Visualization of the PAGE processed protein samples is often achieved through treatment
with a textile dye repurposed for staining proteins. This dye, Coomassie blue, forms non covalent
complexes with proteins. These complexes are believed to be formed due to a mixture of van der
Waals and electrostatic forces. The complex formation stabilizes the negatively charged form of
the dye (Congdon, et. al, 1993).
Circular dichroism (CD) is a technique employed to analyze the secondary structure
(Bulheller, 2007) of proteins through the differential absorption of left and right circularly
polarized light. α-helices and β-sheets give characteristic minima, α-helices at 208 nm and 221
nm while β-sheets display one minimum at 218 nm. Also, unfolded random coil proteins display
a minimum at 198 nm. Fluorescence of tryptophan residues within a polypeptide gives
information relating to the polypeptides tertiary structure. Tryptophan within a polypeptide
absorbs light at 280 nm and emits it at 340 nm, and the intensity of the emitted light relates to the
density of tryptophan residues (Lin and Sakmar, 1996) within a polypeptide and quenching of
residues through exposure to water or ions within an aqueous solution (Lehrer 1971). Quenching
diminishes the intensity of the fluorescence by forming complexes with the chloride ions

5. 5
contained in the buffer solution as the polypeptide unfolds thus allowing analysis of the
polypeptides state of folding.
The secondary and tertiary structures of the protein can be assesed by CD and
fluorescence spectroscopy. Denaturation can be induced in a variety of ways including changes
in temperature and chemical environment (Mainsuradze, 2010). Urea denaturation destabilizes
polypeptide tertiary structure through interfering with internal non-covalent bonds and bonding
to polarized areas of charge (Auton, 2007). This destabilizes tertiary and secondary structure to
the point water and more urea can access the hydrophobic core of the polypeptide.
Figure 1. Ribosomal subunit with EF4 bound in the A site (Gagnon, et. al.,
2014)
Elongation Factor 4/Leading peptidase A is a highly conserved GTPase (Pech, et. al.,
2010; March, Inouye, 1985) that maintains ribosome activity and thus protein production during
inclement cell conditions (Gagnon, et. al., 2014). Unlike most elongation factors EF4 is found on
the periplasmic membrane in E. coli during normal cell conditions instead of the cytoplasm.
Standard state ratios of EF4 are 5/1 membrane:cytoplasm. However during conditions of high
ionic strength and/or low temperature this ratio inverts (Gagnon, et. al., 2014) and by an

6. 6
unknown mechanism releases from the periplasmic membrane into the cytosol to bind with the
ribosomal complex (Figure 1). EF4 has unique back translocase activity, a reversal of the EFG
catalyzed translocase mechanism of the ribosome. EF4’s back translocase activity allows it to re-
mobilize ribosomes that have become immobilized by conformational changes caused by ionic
or low temperature conditions as well as giving EFG a second chance at correct t-RNA
translocation.
EF4 is a competitive inhibitor of EFG and subunits I, II, III, and IV of EF4 share
homology with subunits I, II, III, and V of EFG (Figure 2),(March, Inouye, 1985). However, EF4
has a unique C terminal domain (CTD) instead of EFG’s domain IV that acts as a backstop in the
ribosomal translocation (Qin, et. al., 2006).
Figure 2. EF4 and EFG homology (Qin, et. al., 2006).

7. 7
Materials and Methods
Transformation of expression plasmids into E. coli BL21.
Chemically competent E. coli NiCo21 cells were already on hand as was the unknown
translation factor in plasmid pSV for this experiment. With sterile techniques chemical
transformation was induced using 2 µL (10 ng/µL) of plasmid into 50 µL of the competent E.
coli cells. After the addition of plasmid pSV the pSV/E. coli solution was gently mixed and a
negative control was established by repeating steps with 3 µL of H2O instead of plasmid pSV.
Next the mixtures were placed on ice for approximately 30 minutes after which the
solutions were heat shocked at 42 oC for 60 seconds, then allowed to cool for 3 minutes on ice.
Nine hundred and fifty microliters of Super Optimal broth with catabalyte repression (SOC) was
then added to each solution and the cells were suspended by gently rocking the solutions.
Incubation at 37 oC was then performed for 45 minutes while affixed on a shaking incubator.
Next, 50, 100, and 200 µL of the transformed cells were plated on three Luria bertani broth with
50 µg/mL kanamycin agarose gel plates (LB/Kan-50) respectively and one 200 µL aliquot of the
control solution was plated on the fourth LB/Kan-50 plate. All four plates were then incubated
for approximately 24 hours at 37oC. Kanamycin was added to a concentration of 50 µg/ml to a
flask that contained 50 mL of sterile LB and transferred a single colony from one of our plates to
this kanamycin/LB broth solution using a sterile stick. The seed culture solution was then
incubated on a shaker at 37 oC for another 24 hours.
Bacterial Growth and Recombinant Protein Over-expression.
Sterile techniques were used throughout the following procedure. Six milliliters of sterile
LB-Kan-50 broth was removed from our 3 L flask as an optical OD600 blank. The overnight seed
culture was transferred to a 3 L flask containing 1 L of sterile LB-Kan-50 solution then incubated

8. 8
at 37oC with shaking until it reached an OD600 of 0.5 – 0.6. The E. coli containing solution was
then induced with Isopropyl β-D-1-thiogalactopyranoside (IPTG) to a concentration of 0.75 mM
and quickly placed on a shaker at room temperature for 18-20 hours. After the designated growth
time cells were harvested using centrifugation for 10 minutes at 8281 x g while at a temperature
of 4 OC. The cell pellet was suspended with 25 mL of load/lysis buffer (50 mM Tris-HCl pH 7.5,
150 mM NH4Cl, 10 mM MgCl2, 10 mM imidazole pH 7.0, 15% (v/v) glycerol) and gentle
rocking. The E. coli. suspension was stored at 4 oC.
Cell lysis and Centrifugation
The frozen E. coli pellet was suspended in 25 mL of a load/lysis buffer (50 mM Tris-HCl
pH 7.5, 150 mM NH4Cl, 10 mM MgCl2, 10 mM imidazole pH 7.0, 15% (v/v) glycerol) and
gentle shaking until solution uniformity was observed. Lysis of the cells in solution was achieved
through sonication with a Branson Sonifier 450 for 90 seconds while in an ice bath. The crude
cell lysate was centrifuged at 18,675 x g for 35 minutes at 4oC. The resulting supernatant was
decanted and saved, while the insoluble cell pellet was discarded the supernatant was syringe
filtered in two steps, first, a 5 µm filter, then a 0.45 µL filter.
Dialysis
The filtered supernatant was dialyzed in dialysis buffer (20 mM Tris-HCl pH 7.5, 150
mM NH4Cl, 10 mM MgCl2, 20% (v/v) glycerol) using 25 mm, 2.0 mL/cm, M.W.Co 12-14,000
tubing. The filtered supernatant dialyzed overnight to remove imidazole for future analysis. The
protein aggregated during the dialysis process requiring centrifugation with a Eppendorf
minispin® at 12,000 x g to remove aggregate before all future analysis.
Affinity Chromatography

9. 9
About 5 mL of 50% (Nickel-Nitrilotriacetic acid, Histidine binding resin) Ni-NTA His-
Bind resin equilibrated in load/lysis buffer was poured and settled into a chromatography
column. At a flow rate of approximately 1 mL/min, the centrifuged supernatant was added to the
column and collected as it eluted into a labeled flask. After the lysate flowed through, the column
was rinsed with 10 column volumes of wash buffer #1 (50 mM Tris-HCl pH 7.5, 300 mM
NH4Cl, 10 mM MgCl2, 10 mM imidazole pH 7.0, 15% (v/v) glycerole) and this wash was
collected in a labeled flask. Next, a 10 column rinse and collection was repeated using wash
buffer# 2 (20 mM Tris-HCl pH 7.5, 150 mM NH4Cl, 10 mM MgCl2, 10 mM imidazole pH 7.0,
15% (v/v) glycerol). Last, a 20 tube fraction collection was set up and the protein was eluted at 1
mL/min using the elution buffer (20 mM Tris-HCl pH 7.5, 150 mM NH4HCl, 10 mM MgCl2,
250 mM imidazole pH 7.0, 15% (v/) glycerol) and collected in 2 mL fractions which were
promptly stored on ice.
Quantification
The A280 (absorbance at 280 nm) of each fraction was determined with 2.0 mL aliquot of
each fraction in a quartz cuvette and a UV-VIS HP 8452/8453. A BSA (bovine serum albumin)
standard (1 mg/ml) was measured out to make 50 µL samples at 0, 1, 2, 5, 10, 20 , and 40 µg
respectively in separate microfuge tubes to which 950 µL of Bradford reagent (Coomasie
brilliant blue R250 dye, H3PO4) was added to each tube and incubated for 5 minutes. The
previous steps were repeated with 2, 5, and 10 µL of the unknown protein from the fraction with
the highest concentration. Absorbance readings at 595 nm were then taken for each sample and a
standard curve was constructed from the resulting data to determine concentration of the
unknown through linear fit of the standard curve. Last, the Beer-Lambert Law was applied along

10. 10
with the A280 readings and the protein concentrations from the Bradford Assay to determine the
mass extinction coefficient.
Electrophoresis
Twenty-five mL of 12.5% SDS gel was prepared with (41.6% (v/v) H2O, 12.5% (w/v)
acrylamide, 0.38 M Tris (pH 8.8), and 0.1% (w/v) SDS). After the gel had been mixed, 0.3 mL
of 10& ammonium persulfate-N,N,N',N' (APS) was added along with 0.4 mL of TEMED. The
mixture was poured and then allowed to polymerize after which the gel was placed in the
electrophoresis apparatus and a Tris-glycine buffer (25 mM Tris pH 6.8, 250 mM glycine, 0.1 %
(w/v) SDS, pH 8.3) was added.
Nine samples including wash #1, wash #2 and fractions 2-7 from the affinity
chromatography purification were preapared for electrophoresis. Preperation of the samples was
done with 30 µL of sample and 10 µL of SDS gel-loading buffer (50mM Tris pH 6.8, 100 mM β-
meraptoethanol (βMeSH), 2% (w/v) SDS, 0.1% (w/v) bromophenol blue, 10% (v/v) glycerol).
After mixing the samples were denatured at 100oC for 5 minutes.
Thirty µL samples were loaded onto the gel along with 10 µL of SpectraTM multi-color
broad range protein ladder. The gel electrophoresis was then carried out o completion at 120
volts. After completion the gel was stained using Coomassie brilliant blue stain (250% (w/v)
Coomassie brilliant blue 45% (v/v) methanol, and 10% (v/v) glacial acetic acid) on a shaker table
overnight. Last, the gel was rinsed with tap water and destained for 24 hours by soakin in
methanol:acetic acid (30:10).
Global secondary structure determination of translation factors
Protein samples of ~1.5 mL at ~0.5 mg/mL and ~2.0 mL at ~0.2 mg/mL in dialysis buffer
(20 mM Tris-HCl pH 7.5, 150 mM NH4Cl, 10 mM MgCl2, 20% (v/v) glycerol) were prepared.

11. 11
These samples were used for circular dichroism (CD) spectroscopy and fluorescence
spectroscopy, respectively.
Denaturation of translation factors
Samples of the same volume and concentration as previously dictated with concentrations
of urea from 2 M to 7 M in 0.5 M increments were made to gain unfolded fluorescence
spectroscopy data with a 814 Photomultiplier Detector Spectrophotometer. Data for the buffer
only was also obtained from both CD and fluorescence spectrometry. CD samples were taken
with an Olis DSM10 UV-Vis Spectrophotometer from 40 to 60 Co in 20 increments.
Results
Transformed cells were plated on LB/kan-50 plates in varying amounts. After overnight
incubation on the LB/kan-50 plates two colonies were observed on the 100 µL plate and no
colonies were observed to have grown on the 50 or 200 µL plates. Likewise no colonies were
observed to have grown on the 200 µL control plate. The two colony productivity on the 100 µL
plate gave a transformation efficiency of 1000 CFU/µg.
After plate selection of the transformed E. coli a seed culture of LB broth and kanamycin
was grown using one of the observed colonies, growth to an optical density of approximately 600
was reached after 97 minutes of incubation (Figure 3). Protein over-expression was then induced
using IPTG.

12. 12
Figure 3. Cell culture optical density as a function of time. Initial negative
slope believed to be caused by poor mixing before obtaining the first data
point.
Proteins resulting from an unknown over-expressed translation factor were obtained
along with normal cell proteins through lysing of the expressing cells. The desired protein was
purified by affinity chromatography exploiting the included 6x His-Tag engineered into the
protein resulting in multiple purified fractions (Figure 4), the desired protein eluted early on in
fractions 2-5.
0 25 50 75 100
0.15
0.20
0.25
0.30
0.35
0.40
0.45
0.50
0.55
OpticalDensity
Time (min)

13. 13
1 3 5 7 9 11 13 15 17 19
0
5
10
15
20
25
30
A280
(A.U.)
Protein Fraction
Figure 4. Elution profile of protein absorbance readings of affinity
chromatography purified fractions at 280 nm. Absorbance taken to onfirm and
quantify protein contained within fractions 1-20. Fractions 2-4 were diluted
1/100 but corrected within the graph.
A Bradford assay was preformed to construct a standard curve (Figure 5) to determine the
concentration of the unknown protein samples (Table 1). Using the Bradford assay data and the
Beer-Lambert Law the mass extinction coefficient for the protein was determined to be 2.226
L/G*cm.
Table 1. Analysis of a Bradford protein assay. Absorbance (A.U.) at 595 nm
taken at various volumes. Linear regression was calculated with sample masses
and the BSA standard curve.
Protein Volume
(µL)
Absorbance
(A.U.)
Protein Mass(µg) Protein (g/L)
2 0.0091 2.6 1.3
5 0.15 4.0 0.81
10 0.33 8.9 0.89
20 0.69 19 0.93

14. 14
0 5 10 15 20 25 30 35 40 45
0.0
0.2
0.4
0.6
0.8
1.0
1.2
absorbance(A.U.)
BSA (g)
Linear Fit Parameters:
m = 0.03701
b = 0.03395
R2
= 0.98832
Figure 5. Bradford assay standard curve with linearized fit using bovine serum
albumin. Absorbance (A.U.) at 595 nm. The 40 µg point was excluded to
maintain linearity.
Multiple fractions from Part: C of this experiment were assessed through SDS-
polyacrylamide gel electrophoresis (SDS-Page). Fractions analyzed by polyacrylamide gel are
outlined in (Figure 6) The E. coli lysate, purified for the expressed protein through affinity
chromatography is not completely pure as implicated in (Figure 6) by two bands apparent within
the gel. The band associated with Elongation factor 4 (EF4), through (Figure 6) was

15. 15
Figure 6. SDS-Polycrylamide Gel stained with Coomassie Brilliant Blue.
Lanes from left to right, #1 cell lysate supernatant, #2 crude cell lysate, #3
purified lysate with aggregate, #4 chromatography high speed flow through, #5
chromatography fraction 1, #6 purified cell lysate, #7 Spectratm broad range
protein ladder (kDa), #8 chromatography rinse 2, #9 chromatography rinse 1,
#10 dialysis buffer. Red outline is the EF4 band.
calculated to have a size of 71 kDa, 1.6 kDa (Figure 7) different from EF4's literature value a
2.4% deviation (Shapiro, Maizel, 1967).
#1 #2 #3 #4 #5 #6 #7 #8 #9 #10

16. 16
0.0 0.2 0.4 0.6 0.8 1.0
20
40
60
80
100
MolecularWeights(kDa)
Relative Migration
Log(y) = 2.23 + (-2.77)x1
+ (3.56)x2
+ (-2.2)x3
R2
= 0.994
Figure 7. Relative migration of protein standards from SDS-PAGE against
log10 modified molecular weights. Data points for 260 and 140 kDa excluded
to maintain linearity.
Analysis through fluorescence and CD spectroscopy revealed several properties about
Elongation Factor 4 (EF4). Urea denaturation and subsequent analysis showed that EF4
exhibited a co-operative denaturation with the critical point being at an approximate urea
concentration of 3.4 Molar (Figure 10). Denaturation started at 2.0 M and complete denaturation
occurred at approximately 5.0 M urea.

17. 17
215 220 225 230 235 240
-35
-30
-25
-20
-15
-10
-5
0Ellipticity(mdeg)
Wavelength
40
42
44
46
48
50
52
54
56
58
60
Native
Blank Y
Figure 8. CD ellipticity taken from 313.15-333.15 K, at 215-240 nm. A 205 -
215 nm range was excluded due to loss of continuity.

18. 18
300 325 350 375 400
0
500
1000
1500
2000
2500
Intensity(A.U)
Wavelength
0
0.5
1
2
2.5
3
3.5
4
4.5
5
5.5
6
blank
Figure 9. Fluorescence intensity readings between 0-6 M urea concentrations
from 300-400 nm.

19. 19
0 1 2 3 4 5 6
1600
1700
1800
1900
2000
2100
2200
2300
2400Intensity(A.U.)
Urea (M)
R2
= 0.994
58.7x + 2200
R2
= 0.953
y = -182x + 2688
R2
= -0.755
y = 22.0x + 1600
Figure 10. Fluorescence intensity at 317 nm throughout chemical denaturation
by increasing urea concentration.
Denaturation through temperature during CD analysis showed once again that EF4’s
denaturation was cooperative with a critical point of denaturation being at approximately 321.5 L
(Figure 11), denaturation started at 320 K and complete denaturation occurring at approximately
325 K. CD data were constructed into a van’t Hoff plot (Figure 12) gave us the ΔH and ΔS
which were 863 J/mol and 2.6 J/mol*K respectively. The ΔGo’ was calculated to be 61 J/mol
(Auton, et. al., 2007). EF4 displayed a mixture of both alpha helices and beta sheets within its
secondary structure based on dips at 222 and 218 nm respectively (Chen, et. al. 1974).

20. 20
310 315 320 325 330 335
-30
-28
-26
-24
-22
-20
-18
Ellipticity(mdeg)
Temperature (K)
R2
= 0.542
y = -0.134x + 24.1
R2
= 0.969
y = 1.19x -406.9
R2
= -0.657
y = 0.0625x - 48.6
Figure 11. Ellipticity at 221.8 nm of EF4 throughout thermal denaturation.

21. 21
0.00308 0.00310 0.00312 0.00314 0.00316
-4
-2
0
2
4
6
ln(Keq
)
1/T (K-1
)
R2
= 0.971
y = -103904x + 323.98
Figure 12. Van’t Hoff plot of thermal denaturation between 318 and 325 K as
measured by CD.
Discussion
The results support that transformation occurred with our competent E. coli cells. Due to
colony growth on the kanamycin plates, transformation was confirmed. Furthermore, the
transformation is further supported by the lack of productivity on the control plate implying that
our un-transformed E. coli cells lacked kanamycin resistance and thus were unable to grow on
the control plate.
Productivity of the transformed cells into colony quantity was not as prolific as other
groups and this could be for a couple of reasons. Experimental error within the procedure or the
following of the procedure is a possible source of error leading to lack of colony growth.

22. 22
Another possibility being cell competency was not as advertised by the manufacture nor as
desired. Any or all of these reasons could be why the 50 and 200 µL plates did not produce any
growth. Another possible reason for the small productivity seen is a lack of time in between
transformation and plating on the selecting medium much like the results seen by (Cohen, et al.
1972) with E. coli in their results requiring subsequent incubation after transformation to express
resistance.
First and foremost, based on the overly large mass extinction coefficient no molar
extinction coefficients were determined nor was a postulate as to what translation factor the E.
coli were transformed with was determined. Two trials of the Bradford assay were completed to
obtain a standard curve with a desirable R2 value and multiple issues were addressed to obtain a
reasonable and repeatable A280 value for the unknown protein. Among the issues that were
observed with obtaining repeatable A280 values for the unknown protein included the high
concentration of purified protein, the possible resulting aggregation of the protein due to, or due
to the high protein concentration and experimental error in the application of the HP8452/28453
spectrophotometer #2. Aggregation of the protein is most likely due to the pH of the buffering
solution the protein is suspended in; proteins are often only stable against mis-folding and
possible aggregation over narrow pH ranges around their normal operation conditions. Deviation
from this range leads to mis-folding or even aggregation (Krishnan, et. Al., 2003). Another
possible cause of aggregation could be attributed to the large concentration of protein obtained,
initial aggregation is thought to be represented by first order reactions but subsequent
aggregation after the initial seeding is believed to be represented by higher order reactions and
has been shown to be influenced by increasing protein concentration (Treuheit 2002). Also,
knowing that EF4 is only in the cytoplasm during high ionic or low temperature cellular

23. 23
conditions the buffers used to store it may have been non-ideal for stabilized storage (Chi, et. al.,
2006). Protein aggregation was addressed through centrifugation before any readings were taken
to remove the aggregate; high protein concentration was addressed through larger dilutions for
samples being analyzed and experimental error was addressed through replicates of the
proscribed procedure.
The first indication that lysing and purification had resulted in an unexpectedly large
concentration of protein occurred during the absorbance readings for the affinity eluent fractions
where it was necessary to dilute samples 2-4 (Figure 4) by 1/100 opposed to the 1/20 dilution
recommended in the general procedure to obtain absorbance readings within the
spectrophotometers accepted range. This same indication was observed when attempting to take
the A280 reading for the Bradford Assay when after centrifugation the sample needed a 1/20
dilution to obtain a reading within the accepted range for the instrument.
Third, the Bradford assay gave a standard curve with an R2 linear fit value of 0.98832
with only one outlier point thrown out due to its large deviation from a linear fit. Using this
standard curve the concentration of the unknown protein was determined as outlined in (Table 1)
helped determine the mass extinction coefficient through use of the Beer-Lambert Law A = Ɛbc
to solve for concentration of our unknown: 0.57 µg/µL (Keiichiro 1963)..
SDS-PAGE analysis provided the molecular weight data needed to confirm the
hypothesis that the unknown protein is EF4. The linear model obtained from (Figure 7) gave a
molecular weight with a deviation of 5% from the listed literature value for EF4 (Shapiro,
Maizel, 1967). This degree of error is better than the 10% expected error found by in using SDS-
PAGE for molecular weight determination (Weber, Osborn, 1969). This data along with

24. 24
observed aggregation of the protein compared against the known possibilities and data from
(Walter, et al. 2010) allowed the determination that the expressed protein is EF4.
SDS-PAGE analysis demonstrates that the chromatography purified cell lysate contains
at least one other protein or protein subunit within it. As the lysate containing cell aggregate did
not contain this band, the only difference between the two samples being centrifugation to
remove the solid aggregate it is likely this band is a contaminate. The bands identity being a
protein that eluted similarly to EF4 during the chromatography purification cannot be ruled out
as another possible source of the band. A possible explanation for the streaking near the top of
the gel can be attributed to the volume of sample loaded into the well along with the relatively
high concentration of protein contained within that volume. Lanes #5 and #8-10 showed no
protein contained within the samples which was to be expected as lane #10 was the dialysis
buffer and protein would not be able to cross the dialysis membrane. Lanes 8-9 being the rinses
before the lysate was passed through the chromatography column in Part C (Walter, 2010) of this
experiment, and lane 5 being the first fraction eluted after addition of the lysate into the column
and thus should contain only elution buffer. Lanes #1 and #2 appear identical, being that they are
both lysate from the E. coli cells with the only difference being lane #1 was centrifuged to
remove intact structures and insoluble cell organelles and membrane fragments all of which
would be too large to enter the polyacrylamide gel and result in a band.
Much of the data analyzed through spectroscopy was based on specific wavelengths of
fluorescence and CD, at 317 and 221.8 nm respectively. Raw data graphs of EF4 denaturation
are seen in (Figure 9) and (Figure 9), subsequent calculations based on the data obtained at the
specific wavelengths were applied to obtained thermodynamic values (Mainsuradze, et. al.,
2010). The data obtained through CD were notable in the fact that the range obtained was small

25. 25
and only contained one distinct middle point between the native and denatured state of EF4.
Thus all thermodynamic values obtained from CD analysis are not adequate representative
values of EF4’s true values based on the lack of data points and replicates (Bulheller, et. al.,
2007). Postulates as to why such a week signal was obtained are due to low protein concentration
due to aggregation, aggregation throughout the analysis, or contamination as the protein extract
was shown to be impure during the SDS-PAGE analysis.
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