Enzymes are biological molecules that belong to the family of proteins. They
have an important role in several biochemical functions, serving as natural catalysts that
act to lower the activation energy of specific reactions so that important metabolic
processes can occur at rates sufficient to sustain life (Brocklehurst). Dihydrofolate
reductase (DHFR) is an enzyme that catalyzes the reduction of 7,8-dihydrofolate to
5,6,7,8-tetrahydrofolate (THF). THF, the product of this metabolic pathway, is vital for
the synthesis of certain amino acids, purines and pyrimidines, which are precursors to
macromolecules such as proteins and nucleic acids (Schnell). Therefore, DHFR has an
indispensable role in the growth and proliferation of cells.
The inhibition of DHFR prevents deoxyribonucleic acid (DNA) synthesis by
depleting the building blocks required for their production (purines, pyrimidines)
(Mohebbi). Another result of DHFR inhibition, which leads to cell necrosis, is uracil
accumulation in mitochondrial DNA (mtDNA) (Anderson). Due to these results, DHFR is
an ideal target for antibiotic and anticancer drugs. Anticancer drugs such as metrotrexate
(MTX) and benzamine riboside, and antimicrobial agents such as trimethoprim act as
competitive inhibitors of DHFR(Martorell, Rousell), resulting in the inhibition of DNA
biosynthesis and eventually, cell death. Therefore, further study of the structure and
function of these inhibitors and their interactions with DHFR may lead to the discovery
of new antibiotic/anticancer drugs as well as increased knowledge and improved
efficiency of existing ones. Due to the promising results of DHFR inhibition, it has been
widely described as the 'enzyme of choice for all seasons and almost all
reasons'(Sharma).
Although promising, DHFR inhibition has a major setback. The clinical efficacy
of normally successful compounds can be compromised by resistance arising through
mutations at various sites on the enzyme. Certain mutations of DHFR can therefore
render several anticancer/antimicrobial compounds useless (Yuthavong).
The objective of this research project is to over-express, purify and crystallize
DHFR in order to screen for new inhibitor compounds to discover new marketable
anticancer/antimicrobial compounds (Vulcu). Techniques required for the overexpression, purification, characterization and finally crystallization of DHFR will be
described, as they will be vital towards producing large amounts of functional DHFR.
High quantities of high-quality DHFR can enable the use of screening methods (eg. highthroughput screening of compound collections) for inhibitors that may be novel and
increasingly effective antibiotics or anticancer drugs (Vulcu). Increased knowledge of
effective DHFR-inhibitor compounds would be invaluable to the field of oncology and
humankinds ongoing battle with cancer.
Flowchart of Experiments
Figure 1. Flow chart of experiments involved in the determination of novel inhibitors of DHFR. (1) Gene
cloning was performed on folA to generate a population of Escherichia coli BL21-DE3 cells containing a
pET28b vector including the gene of interest. (2) Restriction digests with PvuI (single) and NdeI & XhoI
(double) were performed to test for the presence of folA within the pET28b vector. (3) BL21-DE3 cells are
utilized because of lon- and omp-protease deficiency, protecting expressed DHFR from proteolytic
cleavage. (4) A Kanamycin medium is utilized to select for kanamycin-resistant cells that were successfully
transformed with pET28b-folA, which confers kanamycin resistance. (5) Induction with IPTG removes the
lac repressor from the lac operon and allow for translation of T7 RNA Polymerase, which will transcribe
folA. (6) Cells are lysed to extract expressed DHFR. (7) DHFR is present in the supernatant because it is a
cytosolic protein. (8) The His(6)-tag creates high affinity to the Ni-NTA column (purification), therefore
DHFR elutes last when an imidazole-based buffer is used. (9) (10) Determination of protein concentration
and relative purity of protein sample in elution fractions. (11) The Sitting Drop Method was employed for
the generation of crystals via precipitation out of solution using salt. (12) Screening performed to create a
collection of successful wild-type DHFR inhibitors. (13) Comparative Molecular Field Analysis (CoMFA)
can optimize the inhibitory efficiency of molecules based on successful DHFR inhibitors. (14) HighThroughput Screening of derived compounds on crystallized DHFR to determine strongest inhibitors. Other
factors (ie. Lipinskis* Rule of 5) taken into consideration to determine most useful compounds. (15) Most
suitable inhibitors are administered as drugs in clinical trials.
the bed. 3 mL of wash buffer (100 mM NaH2PO4, pH 8.0, 300 mM NaCl, 20mM
imidazole) was then gently pipetted onto the side of the resin bed using a plastic transfer
pipette. 3 mL of wash buffer was applied again, and each time one 1 mL aliquot of each
wash was collected in a 1.5 mL microcentrifuge tube which was labeled frozen at -20C
for future use. The column was then drained into the waste beaker until approximately 1
mm of buffer remained above the bed. 5 mL of elution buffer (100 mM NaH2PO4, pH
8.0, 300 mM NaCl, 250 mM imidazole) was added onto the centre of the column and five
1 mL fractions were collected into 1.5 mL microcentrifuge tubes, which was labeled and
frozen at -20C for future use.
Protein Quantification
Four 1 mL serial dilutions (1:2) were made from the stock Bovine Serum Albumin (BSA)
standard (1 mg/mL). 100 L of each dilution was added into clean, dry test tubes in
triplicate (15 tubes in total). 100 L of undiluted and 1:2 diluted flow-through solutions
respectively were added to two test tubes in triplicate (6 tubes in total). 100 L of
undiluted and 1:2 diluted elution fractions 1 and 2 respectively were added to 4 test tubes
in triplicate (12 tubes in total). 100 L of the dilution buffer was pipetted into a clean, dry
test tube (reagent blank). 5 mL of diluted protein-assay dye reagent (Bio-Rad) was added
to the sample tubes containing the BSA protein standards, flow-through, elution fractions,
and reagent blank respectively. After addition of the dye, the tubes were immediately
inverted to mix the contents. Tubes were then left undisturbed for 5 min. The absorbance
of the solution at 595 nm (A595) was then measured on the Spec20 (Thermoscientific),
using the reagent blank as a measure for zero absorbance. The A595 was then measured
for each tube, from lowest to highest protein concentration, using the Spec20. The
absorbencies of the aliquots of each sample were also measured. The Spec20 tubes were
then cleaned with water. Excess Bradford reagent and waste were collected in bottles.
Determination of Relative Protein Sample Purity
The Mini-PROTEANTGX pre-cast gel was prepared (via the protocol outlined in the
catalogue by Bio-Rad*) by McMaster University, Department of Biochemistry and
Biomedical Sciences. Protein samples were prepared while waiting for the gel to
polymerize. Using 1.5 mL Eppendorf tubes, 15 L of all samples obtained in last section
in addition to 15 L of 2X SDS-PAGE loading buffer (Bio-Rad: 2.96 mL distilled water,
40 L 0.5 M EDTA-Na2, 2 mL Glycerol, 770 mg DTT, 4 mL 10% SDS, 2 mg
bromophenol blue in total volume of 10mL) were aliquotted into each tube. Protein
fractions were kept on ice during use, then returned to the -20C freezer. The samples
were boiled for 2-3 minutes. The tubes were then centrifuged for approximately 2
seconds. The order of SDS-PAGE gel loading was recorded. Approximately 23 L of
sample was loaded per well. The gel running apparatus (Bio-Rad) was turned on to 175 V
and the gel was run for approximately one hour. When the dye front reached the very
bottom of the separating gel, the voltage was turned to zero and the power supply was
turned off. The electrode assembly was removed and unclamped, and the gel sandwich
was gently removed. The gel was removed using a gel releaser. The gels were then
placed in a Coomassie staining solution (Bio-Rad: 50% V/V methanol, 5% V/V acetic
acid, 0.1% V/V Coomassie Brilliant Blue G-250) in a fumehood and placed on a gel
rocker overnight. The gels were then destained using a destaining solution (10% V/V
acetic acid, 5% V/V methanol), dried and scanned by McMaster University, Department
of Biochemistry and Biomedical Sciences.
Crystallization of His(6)-DHFR
DHFR crystallization was accomplished utilizing a SaltRx protein crystallization
reagent incorporating the Sitting Drop Method (Hampton Research). 166.7 mg/mL
lysozyme dissolved in Buffer A (sodium acetate, pH 4) and SaltRx Reservoir Buffer
(0.1 M Sodium Acetate, pH 4.8, 6.5% W/V NaCl) were pipetted onto the sample wells on
unit 1 to 3 of the Intelli-Plate 24-4 well. Elution fractions 1, 2, and 3 (with reservoir
buffer and 0.17 M Buffer A) were added to the sample wells on unit 4, 5, 6 of the plate
respectively. Protein crystals were visualized by Nikon SMZ1500 microscope (1.5X
magnification), scanned by Lumenera Infinity 2 camera and Infinity Analyze camera
software.*
RESULTS
kb
pET28b-fola
6000
pET28b
folA
500
Lane
Figure
2.
Agarose
Gel
Electrophoresis of Digested PCR
Amplicon and pET28b. Figure 2
contains a 1% agarose gel stained
with GelRed Nucleic Acid Gel
Stain containing the PCRamplified folA gene and pET28bfolA plasmid vector digested with
restriction enzymes NdeI and
XhoI. The gel was run at 150V at
22C. Lane 1 contains the 1
kilobase DNA ladder. Lane 2
contains the folA amplicon. Lane
3 contains the uncut pET28b-folA
plasmid. Lane 4 contains the
pET28b-folA plasmid cut with
restriction enzymes NdeI and
XhoI. The gel was visualized
under UV light and photographed
using the Kodak Gel Logic 100
camera.
kb
6000
pET28b
PvuI
Fragment 1
PvuI
Fragment 2
500
250
folA
RNA
Lane
Figure
3.
Agarose
Gel
Electrophoresis of single- and
double-digested
pET28b-folA
gene plasmid. Figure 3 contains
a 1% agarose gel stained with
GelRed Nucleic Acid Gel
Stain containing loaded samples
of the pET28b-folA plasmid
digested
with
restriction
enzymes PvuI and NdeI & XhoI
respectively. The gel was run at
150V at 22C. Lane 1 contains
the 1 kilobase DNA ladder. Lane
2 contains the uncut pET28bfolA plasmid. Lane 3 contains
the
pET28b-folA
plasmid
double-digested with restriction
enzymes NdeI and XhoI. Lane 4
contains
the
pET28b-folA
plasmid single-digested with
restriction enzyme PvuI. The gel
was visualized under UV light
and photographed using the
Kodak Gel Logic 100 camera.
0.9
0.8
0.7
0.6
Induction
with IPTG
Log (exponential
phase)
0.5
0.4
0.3
0.2
Lag phase
0.1
0
0
50
100
150
200
Time (minutes)
250
300
350
Figure 4. Bacterial Growth Curve of E. Coli BL21-DE3 Cells Induced with IPTG. Figure 4 displays a
growth curve of the harvested E. coli cells bearing the pET28b-folA plasmid. The Y-axis displays the
optical density of the sample at a wavelength of 600 nm (measured by Spec20 spectrophotometer). The Xaxis displays the time elapsed in minutes. An arrow indicates the point of induction with IPTG (230
minutes). The lag phase, log (exponential) phase, and onset of stationary phase are also labelled.
0.75
0.7
y = 0.1527x + 0.3465
R = 0.9933
0.65
0.6
0.55
0.5
0.45
0.4
0.35
0
0.5
1
1.5
2
Concentration of BSA (mg/mL)
2.5
Figure 5. Standard absorbance curve for Bovine Serum Albumin (BSA) at Varying Concentrations utilizing
the Bradford Assay technique. Figure 5 displays a curve reflecting the absorbance values of BSA standards
at 595 nm, at varying concentrations of BSA in a Bradford assay. The absorbance values were measured
using the Multiskan Ascent Plate Reader (Thermo-Scientific). The Y-axis denotes the absorbance average
of the BSA standard (measured in triplicate). The X-axis denotes the concentration of BSA measured in
mg/mL. The linear equation represents the relationship between BSA absorbance and BSA concentration.
The closeness of R2 value to 1 indicates the strength of the correlation between the linear equation and data
points, with a value of 1 representing perfect correlation. The standard deviation of each sample is
indicated by the error bars shown on the curve.
Dilution
Stock BSA
1
2
3
4
Absorbance 1
0.593
0.638
0.421
0.372
0.380
Absorbance 2
0.565
0.596
0.434
0.373
0.343
Absorbance 3
0.790
0.548
0.461
0.382
0.361
Average
0.649
0.594
0.439
0.376
0.361
Table 1. Absorbance values of serial dilutions of BSA. Table 1 displays the various absorbance values
measured for the stock BSA solution and each of the serial dilutions, measured using the Multiskan Ascent
Plate Reader (Thermo-Scientific). The absorbance of the blank sample was adjusted to 0 and all other
average absorbance values were adjusted relative to the absorbance of the blank sample.
kDA
245
180
135
100
75
63
48
35
25
20
DHFR
17
11
Lane
10
Figure 6. SDS-PAGE gel of Ni-NTA Affinity Column Elution Fractions. Sodium dodecyl sulfate
polyacrylamide gel electrophoresis (SDS-PAGE) was run for approximately 1 hour at 175 V, 22C after
eluted fractions were obtained from nickel affinity column chromatography. A protein molecular weight
marker (Gene DireX) is present in Lane 2 in order to measure the molecular weight (size) of protein
samples in other lanes. Lane 1 contains the cell lysate. Lane 3 was contains the flow through. Lanes 4 and 5
contain the first and second washes respectively. Lanes 6, 7, 8, 9, 10 contain elution fractions one, two
three, four and five respectively.
Figure 7. Photographs of (A) lysozyme crystals and (B) DHFR crystals formed via the SaltRxSitting
Drop method. Protein crystallization of both lysozyme (7a) and DHFR (7b) was accomplished utilizing the
SaltRxSitting Drop method at -4C for one week. The lysozyme was used as a positive control to which
DHFR crystallization could be compared. Photographs were taken of wells C3 (lysozyme) and C5 (DHFR)
of the Intelli-PlateTM using a Nikon SMZ1500 microscope (1.5X magnification), Lumenera Infinity 2
camera and Infinity Analyze camera software.