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'

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= 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).

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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).

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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

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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

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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).

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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

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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

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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

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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.

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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.

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0 25 50 75 100

0.15

0.20

0.25

0.30

0.35

0.40

0.45

0.50

0.55

Op

tica

l De

nsi

ty

Time (min)

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.

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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 1.35 0.15 4.0 0.8110 0.33 8.9 0.8920 0.69 19 0.93

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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

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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

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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.

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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.

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Figure 9. Fluorescence intensity readings between 0-6 M urea concentrations from 300-400 nm.

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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).

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Figure 11. Ellipticity at 221.8 nm of EF4 throughout thermal denaturation.

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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.

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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

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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

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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

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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.

References

Auton, M., Holthauzen, M., and Bolen, D. (2007). Anatomy of energetic changes accompanying urea-induced protein denaturation. Proceedings of the National Academy of Sciences. Vol. 104(39). 15317-15322.

Bulheller, B., Rodgerb, A., Hirst, J. (2007). Circular and linear dichroism of proteins. Physical Chemistry Chemical Physics. Vol. 9. 2020-2035.

Candau, F., Leong, Y.S. and Fitch, R.M. (1985) Kinetic study of the polymerization of acrylamide in inverse microemulsion. Journal of Polymer Science: Polymer Chemistry Edition, Vol. 23:193-214.

Cao P, Wang L, Zhou G, Wang Y, Chen Y. (2014). Rapid assembly of multiple DNA fragments through direct transformation of PCR products into E. coli and Lactobacillus. Plasmid. Vol. (76) 40-46.

Finer, E.G. Flook, A.J. Hauser, H. (1972) Mechanism of sonication of aqueous egg yolk lecithin dispersions and the nature of the resultant particles. Biochimica et Biophysica Acta, Lipids and Lipid Metabolism. Vol. 260(1), 49-58.

Chen, Y-H., Yang, J., Chau, K. (1974). Determination of the helix and β form of proteins in aqueous solution by circular dichroism. Biochemistry. 13(16). 3350-3359.

Chi, Eva Y. Krishnan, Sampathkumar. Randolph, Theodore W. Carpenter, John F. (2003). Physical stability of proteins in aqueous solution: Mechanism and driving forces in nonnative protein aggregation. Pharmaceutical Research, Vol. 20(9), 1325-1336.

Cohen, Stanley. Chang, Annie. Hsu, Leslie. 1972. Nonchromosomal antibiotic resistance in bacteria: Genetic transformation of Escherichia coli by R-Factor DNA. Proceedings of the National Academy of Sciences, Vol. 69(8), 2110-2114.

26

Congdon, RW. Muth, RW. Splittergerber, AG. (1993) The binding interaction of Coomassie blue with proteins. Analytical Biochemistry, 213(2), 407-413.

Gagnon G., Matthieu. Lin, Jinzhong. Bulkley, David. Steitz A., Thomas. (2014) Crystal structure of elongation factor 4 bound to a clockwise ratcheted ribosome. Science, Vol.345(6197), 684-687.

Keiichiro, Fuwa. Valle, B. L. 1963. The physical basis of analytical atomic absorption spectrometry. The pertinance of the Beer-Lambert law. Analytical Chemistry. Vol. 35(8), 942-946.

Lehrer, S. (1971) Solute perturbation of protein fluorescence. Quenching of the tryptophyl fluorescence of model compounds and of lysozyme by iodide ion biochemistry. Biochemistry. Vol. 10 (17). 3254–3263

Lin, S. and Sakmar, T. (1996) Specific Tryptophan UV-Absorbance Changes Are Probes of the Transition of Rhodopsin to Its Active State Biochemistry. Biochemistry. Vol. 35 (34). 11149–11159

Mainsuradze, G., Liwo, A., Otdziei, S., Scheraga, H. (2010) Evidence, from simulations, of a single state with residual native structure at the thermal denaturation midpoint of a small globular protein. Journal of the American Chemical Society. Vol. 132(27). 9444-9452

March, P. E. Inouye, M. (1985). GTP-binding membrane protein of Escherichia coli with sequence homology to initiation factor 2 and elongation factors Tu and G. Proceedings of the National Academy of Sciences of the United States of America. Vol. 82(22), 7500-7504

Matzke, A.J. Chilton, M.D. (1981) Site-specific insertion of genes into T-DNA of the Agrobacterium tumor-inducing plasmid: an approach to genetic engineering of higher plant cells. Journal of Molecular and Applied Genetics. Vol. 1(1):39-49.

Pech, Markus. Karim, Zhala. Yamamoto, Hiroshi. Kitakawa, Madoka. Qin, Yan. Nierhuas H., Knud. (2010). Elongation factor 4 (EF4/LepA) accelerates protein synthesis at increased Mg2+ concentrations. Proceedings of the National Academy of Sciences of the United States of America. Vol. 108(8), 3199-3203.

Qin, Yan. Polacek, Norbert. Vesper, Oliver. Staub, Eike. Einfeldt, Edda. Wilson N., Daniel. Nierhaus H., Knud. (2006). The Highly Conserved LepA Is a Ribosoomal Elongation Factor that Back-Translocates the Ribosome. Cell Vol. 127(4), 721-733.

Reynolds, Jacqueline A., Tanford, Charles. (1970) The gross conformation of protein-sodium dodecyl sulfate complexes. The journal of Biological Chemistry, Vol. 245,5161-5162.

Scheich, Christoph. Sievert, Volker. Bussow, Konrad. (2003) An automated method for high-throughput protein purification applied to a comparison of His-tag and GST-tag affinity chromatography. Bio Med Central, Biotechnology Vol. 3:12

27

Shapiro AL, Viñuela E, Maizel JV Jr. (1967). Molecular weight estimation of polypeptide chains by electrophoresis in SDS-polyacrylamide gels. Biochemical and Biophysical Research Communications. Vol. 28(5): 815–820.

Treuheit, Michael J. Kosky, Andrew A. Brems, David N. 2002. Inverse relationship of protein concentration and aggregation. Pharmaceutical Research, Vol. 19, 511-516

Weber, Klaus. Osborn, Mary. (1969) The reliability of molecular weight determination by dodecyl sulfate-polyacryalmide gel electrophoresis. The Journal of Biological Chemistry. Vol. 244, 4406-4412.

Walter, D. Justin. Littlefield, Peter. Delbecq, Scott. Prody, Gerry. Spiegal, P. Clint. (2010) Expression, purification, and analysis of unknown translation factors from Escherichia coli: a synthesis approach. Biochemistry and Molecular Biology Education, Vol. 38(1), 17-22.

Weber, Klaus. Osborn, Mary. (1969). The reliability of molecular weight determinations by dodecyl sulfate-polyacrylamide gel electrophoresis. The journal of Biological Chemistry. Vol. 244, 4406-4412.