Carleton University
Formal Report
Course #: BIOC-3104
Experiment #: 2
Expression, Purification and Characterization of a Recombinant Protein
– Taq polymerase
Day submitted: 2022/03/07
Abstract
The aim of this experiment is to extract and purify the Taq polymerase enzyme
expressed by E.Coli expressing system. Also, the activity of the enzyme acquired is
detected by PCR and quantified by RT-qPCR. Desalting, size exclusion
chromatography are used to purify the enzyme samples. SDS-PAGE and western blot
are used to validate the purity and the identity of the enzyme obtained. In addition,
Bio-Rad assay is used in this lab to quantify the concentration of protein samples
acquired. In terms of the results, the protein concentration of 2022 Taq sample is
detected to be 14.7 ± 12.7 mg/mL, and for 2021 Taq sample is 69.4 ± 5.46 mg/mL.
The efficiency of the Taq polymerase was detected to be 79.2% by RT-PCR. The
SDS-PAGE indicated the Taq polymerase extracted in this lab had a MW of 106.32 ±
0.17 kDa, which had a 13.0 % error compared with theoretical one, 94.05 kDa. The
Western Blot and PCR techniques employed validated the identity and activity of Taq
polymerase. The Taq enzyme purified is generally pure, where 2022 sample is more
pure than the 2021 sample. Excepting the 2022 Thursday’s sample, all the Taq
enzyme samples were desalted well because there were no water spots appeared on
the gel. To sum up, this experiment is successful, however, some aspects can still be
further optimized, such as steps of bacteria cells lysis, protein extraction, and Western
Blot technique.
Introduction
The purpose of this experiment is to purify and extract the Taq polymerase enzyme
expressed in E.Coli BL21(DE3). Additionally, the acquired enzyme's activity is
identified and quantified using PCR and RT-qPCR. Purification of enzyme samples is
accomplished using desalting and size exclusion chromatography. SDS-PAGE and
western blot are used to confirm the enzyme's purity and identification. Additionally,
this lab utilises a Bio-Rad test to determine the concentration of protein samples
obtained.
Taq polymerase is a kind of DNA polymerase that works well under a high
temperature. It has an optimal working temperature at 75-80 oC, where this advantage
has been widely applied to PCR amplification. The taq polymerase has a function of
5’to 3’ exonuclease, to proofread the DNA sequence, preventing from mismatch.
However, it lacks an ability of proofreading the DNA sequence synthesized in a
direction of 3' to 5’ [3], leading to an error probability of nucleotide mismatch for
every 3×104 and 3×106 times of nucleotide extension [4]. Rather than 75-80 oC,
another article has reported a nucleotide extension rate of 2-4 kilobases per minute at
its optimal temperature of 72 oC [5]. The optimal buffer solution has been reported to
be pH 9.4, and 10-55 mM KCl and 2-3 mM MgCl2 [3]. Also, an optimal buffer with
pH 8.3, with50 mM KCl and 1.5 mM MgCl2 has been reported [5]. Using other
divalent metal ion instead of Magnesium will decrease the enzyme acitivity [6].
Furthermore, too high concentration of monovalent metal ion has been reported to
inhibit the activity of the Taq polymerase, for example higher than 100 mM [7,8].
Many factors can affect protein stability under heat shock condition.
Representative factors involve amino acids distribution, polar surface area, hydrogen
bonds and salt bridge, helical content etc [9]. The enzyme of interest in this lab, Taq
polymerase, has a robust thermostability, which is initially isolated from an extremely
thermophilic bacteria called Thermus aquaticus. The high thermostability of Taq
polymerase can be primarily attributed to its very negative folding free energy,
making the proteins very stable. Furthermore, lower entropic folding penalty has also
contributed to its thermostability, which means it will suffer less impact on its folding
free energy when the temperature turns to a higher one. Therefore, its denatured
structure under heat shoc will have much smaller changes on its structure, dynamic or
desolvation, comparing to others [10].
Actually, before the PCR technique launched, the genome-relevant research
was a very struggling work, since whole genome was always studying simultaneously.
The PCR technique invented latter on has remarkably promote molecular biology.
PCR technique can be traced back to 1850s. Kornberg’s research group has
discovered and validated the DNA can be replicated itself under the involvement of
DNA polymerase in E.Coli in 1957 [11]. In 1969, Brock and Freeze found
thermostable bacteria Thermus aquaticus from hot springs of Yellowstone National
Park and the Taq polymerase was isolated in the following year [12]. These two
explorations have built a solid foundation for PCR technique. Until 1986, Mullis
introduced the concept of using thermostable DNA polymerase to replicate DNA [13].
Their research group referred to the idea reported by Sanger’s research group that use
primer to control the replication end site of DNA sequence [14] .
Experimental Protocol
A. Culturing, Expression and Lysis
A flask containing 125 μL E. Coli BL21(DE3) cell culture, 600 mL of Luria
Broth (LB) media, with 0.01 % (w/v) ampicillin was incubated for 24h at 37 oC in
New Brunswick Scientific Incubator. The culture was diluted to an absorbance of 0.8,
then 68 mg of IPTG was added into the culture and incubated for 18h at 37 oC in the
incubator.
The cells were obtained by 15 minutes of 4000 xg centrifuge by Eppendorf
Centrifuge 5810 followed by discarding the supernatant. To lyse the bacteria cells, 20
mL of buffer A and buffer B were prepared and stored at 4 oC. Buffer A involved 50
mM Tris HCl (pH 7.9), 50 mM Dextrose, 1 mM EDTA, and ddH2O to the volume.
Buffer B involved 10 mM Tris HCl (pH 7.9), 50 mM KCl, 0.5% Tween 20, 0.5%
NP-40, 1 mM PMSF, 1 mM EDTA and ddH2O to the volume.
The pellet obtained by centrifuge was resuspended in 6 mL of Buffer A by
blowing the mixture using pipette. Repeated this again and collected totally 12 mL
cell suspension in a new centrifugetube. Centrifuged it for 15 minutes at 4000 xg by
Eppendorf Centrifuge 5810 followed by discarding the supernatant. Resuspended the
pellet by 6 mL of Buffer A containing 24 mg of lysozyme, following by 15 min
incubation. Then, added 6 mL of Buffer B to cell suspension and incubated it for 1h at
80 oC in New Brunswick Scientific Incubator.
Collected the suspension and centrifuged it at 16000 xg for 10 min at 4 oC, by
using Thermo Scientific Sorvall RC 6+ centrifuge. The supernatant was collected in a
new tube followed by adding 3.6g of (NH4)2SO4, incubating for 10 min with shaking
at r.t. in the incubator. Afterward, centrifuged it at 16000 xg for 10 min at 4 oC,
discared supernatant. Resuspended it by 1 mL of Buffer A.
B. Desalting by size exclusion chromatography
The column was prepared as manufacturers instructions. The column then was
operated 1000 xg for 2 min using Thermo Scientific Sorvall - Legend Micro 21R.
Discarded the tube and the column was then placed in a new tube. An aliquot of
250 μL supernatant was added into the column and operated 1000 xg for 4 min, then
repeated this again by adding another aliquot of 250 μL supernatant. Discarded the
column and collected the total 500 μL of Taq sample.
C. Bio-Rad Protein concentration Determination
The standard solutions was prepared as manufacturers instructions. In a
transparent microplate, pipetted 10 μL of each standard solutions and 50x diluted
2022 Taq sample and 25x diluted 2021 Taq sample into separate microplate wells, in a
manner of triplicate. Then, 190 μL of Bio-Rad Dye Reagent (Cat. No. 500-0006) to
each well, mixed by vortex. After incubated for 10 min at r.t., the absorbance of
samples and standards at 595 nm were detected by using BioTek Epoch2 microplate
reader.
D. SDS-PAGE & Western Blot
A 10% stained-free SDS-contained acrylamide gel was prepared following the
instruction of BIO-RAD stain free gel. To each new tube, 15 μL of each diluted
samples and 15 μL of Laemmli buffer with beta-mercaptoethanol was added. The
tubes were operated boiling water bath for 3 min and then 5min on the ice. After
loading 7 μL protein marker to 1 well, Precision Plus Protein Unstained Standards
(Cat. # 161-0363), 20 μL of prepared samples were loaded to the wells sequentially.
The gel were ran for 23 minutes under 250V. Finally, the BioRad ChemiDoc XRS+
system with ImageLab software were used to image and visualize the gel.
To operate Western Blot, two immobilon-P transfer membrane (0.45 mm pore
size) and two blotting paper (Whatman paper) that are slightly smaller than gel were
prepared. The membrane was immersed in 100% methanol to wet it, then transferred
membrane to immerse in MilliQ water for few minutes. Removed excess transfer
buffer by inverting the cassette base and placed the cassette lid on the base. Then the
sanwiches was set up following the Trans-Blot Turbo Transfer System operation
manual provided by BIO-RAD. Then, transferred the proteins to the membrane at
30 V using Turbo Transfer System. The membrane was then placed in TBST buffer
(pH 7.6) and stored in 4 oC fridge. In another day, the membrane was blocked in 1 g
Carnation nonfat dry milk (5% w/v) plus 10 ml TBST Buffer (pH 7.6). Then added
12.5 μL of primary antibody, Anti-Taq Monoclonal Antibody (8C1) (eEnzyme:
MA-029-0250), to the 10mL blocks solution, giving a dilution factor of 800x. After
gently shaking for 1h at r.t., washed the solution in TBST Buffer (pH 7.6) for 3×15
minutes at r.t.. Then secondary antibody, Peroxidase-conjugated AffiniPure Goat
Anti-Mouse IgG (H+L) (Jackson ImmunoResearch Laboratory Inc.: 115-035-003),
was added to new block solution, following by shaking for 1h at r.t.. Washed the
solution again using TBST Buffer (pH 7.6) for 3×15 minutes at r.t.. Mixed 1 mL of
Enhanced Luminol Reagent with 1 mL of Oxidizing Reagent of the Renaissance
Western Blot kit (NEN Life Science Products, Cat. No.NEL 101). Cleaned the edge of
blots using tweezers with a kimwipe. A 2 mL of Renaissance mix was added to the
blot, then BioRad ChemiDoc XRS+ system with ImageLab software were employed
to image and visualize the gel.
E. RT PCR
A total 300 μL of Master mix for 15 PCR reactions was prepared following the
standard protocol. Here, the dye used for quantifying DNA content was EvaGreen
Dye (Biotium: 31000). To five microcentrifuge tubes, 48 μL of Master Mix, 6 μL of
Friday Taq polymerase sample, and 6 μL of 5 dilutions of the template DNA (0.1, 0.01,
0.001, 0.0001, 0.00001 ng) were added. After gently vortexing the tubes, transferred
20 μL of each sample to 96-well RT PCR plate, in a manner of triplet. Then sealed the
plate with a compatible sealer, then put it in the Bio Rad CFX Connect Real-Time
PCR detection system with operating standard protocol.
F. PCR
A total 100 μL of Master mix for 4 PCR reactions was prepared following the
standard protocol. To a new microcentrifuge(PCR) tube, 22.5 μL of PCR Master Mix,
and 2.5 μL of experimental Taq polymerase sample were added. An old Taq
polymerase sample was prepared in another new microcentrifuge. These two tubes
were operated standard protocol of PCR reaction using Bio Rad T100 Thermal Cycler.
After the PCR reactions accomplished, the PCR products were analyzed by 1%
agarose gel electrophoresis at 150V for about 20 min. In this case, 20 μL of each PCR
reaction product, and 4 μL of DNA loading dye, Gel Red dye. A 10 kb ladder (Bio
Basic Inc. Cat. No M101R-1) were added to each wells. The BioRad ChemiDoc
XRS+ system with ImageLab software then were used to image and visualize the gel.
Results
1. Bio-Rad
Table 1: Calculated Taq polymerase samples concentration by using the BSA protein
concentration standard curve.
Sample
2022 Taq Sample
2021 Taq Sample
Protein
Concentration
(mg/ml)
0.0147 ± 0.0127
0.1388 ± 0.0109
Dilution factor
Undiluted protein
Concentration
(mg/mL)
14.7 ± 12.7
69.4 ± 5.46
1000
500
0,400
Absorbance, at 595 nm
0,350
0,300
0,250
0,200
0,150
0,100
y = 1.3151x
R2 = 0.9861
0,050
0,000
0
0,05
0,1
0,15
0,2
0,25
0,3
-0,050
BSA Concentrations (mg/mL)
Figure 1. Standard curve of BSA protein concentration vs absorbance at 595 nm using
Bio-Rad protein assay regarding standard BSA solutions. The dye reagent kit used was
Bio-Rad Dye Reagent Cat. No. 500-0006. The dye reagent used in this assay was operated 5x
dilution, and the absorbance was measured at 595 nm, by using BioTek Epoch2 microplate reader.
Table 2: LINEST function regarding standard curve of BSA protein concentration vs
absorbance at 595 nm.
1.315063437
0
Slope
Intercept
e(Slope)
R^2
F
ss reg
e(Intercept)
se(y)
df
ss resid
0.035948022
0.995536607
1338.268938
0.312371402
#N/A
0.01527791
6
0.001400487
The BioTek Epoch2 microplate reader provided all the absorbance data. By making it
relevant to specific protein concentration, a standard curved calculated by EXCEL
was acquired as y=1.351x, R2=0.9861.
Sample calculation

2022 Taq Sample:
0.337  0.328  0.350
 0.3383
3
Diluted Taq sample absorbance:
Error:
(0.337  0.3383) 2  (0.328  0.3383) 2  (0.350  0.3383) 2
 0.0110(mg / mL)
3 1
Blank absorbance:
0.331  0.306  0.320
 0.319
3
Error:
(0.331  0.319) 2  (0.306  0.319) 2  (0.320  0.319) 2
 0.013
3 1
Abs Avg -blank: 0.3383-0.319=0.01933
Error:
0.0132  0.0112  0.01671
0.01933
Diluted Taq sample concentration: 1.3151
2
 0.0147(mg / mL)
2
 0.03595   0.01671 
 
  0.0127(kDa)
  1..3151   0.01933 
  0.0147  
Error:
Total dilution factor is: 1000x.
Thus, undiluted Taq sample concentration: 0.0147 1000  14.7(mg / mL)
Error on the Taq sample concentration:
0.0127 1000  12.7(mg / mL)
2. SDS-PAGE
3
log(MW), kDa
2,5
2
1,5
1
y = -1.4132x + 2.3055
R2 = 0.9437
0,5
0
0
0,2
0,4
0,6
0,8
1
1,2
Relative Front
Figure 2. Relationship between Log(Molecular Weight) versus electrophoretic mobility (Rf) for protein
marker that is shown as the ladder band. The LINEST function and the R2 is calculated by EXCEL,
and are both shown in the attached EXCEL document.
Table 3: LINEST function regarding relationship between Log(molecular weight) versus
electrophoretic mobility (Rf) for protein marker.
Slope
Intercept
1.315063437
0
e(Slope)
e(Intercept)
0.035948022
#N/A
R^2
se(y)
0.995536607
0.01527791
F
df
1338.268938
6
ss reg
ss resid
0.312371402
0.001400487
The ImageLab software provided all the electrophoretic mobility data. The standard
curve of marker represented by relationship between Log(Molecular Weight) versus
electrophoretic mobility (Rf), was calculated by EXCEL as y= -1.4132x+2.3055,
R2=0.9437.
Figure 3. SDS polyacrylamide gel electrophoresis (SDS-PAGE) pattern of Taq polymerase protein
samples: lane 1-2021 25x diluted Taq polymerase sample, lane 2-2022 Thursday 5x diluted Taq
polymerase sample, lane 3-2022 Friday 10x diluted Taq polymerase sample, lane 4-Wednesday 5x
diluted Taq polymerase sample, lane 5- Marker (MW range of 250-10 kDa), lane 6-standard Taq
polymerase sample. The experimental Taq polymerase samples were extracted from E.Coli
BL21(DE3). The proteins sample were ran on 10% stained-free SDS gel, and operated for 23 minutes
under 250V. The ladder protein used was Precision Plus Protein Unstained Standards (Cat. # 161-0363).
The BioRad ChemiDoc XRS+ system with ImageLab software were used to image and visualize the
gel. The detailed information for all bands are calculated and are shown in the EXCEL file attached.
Table 4: Summary of the calculated results of Taq polymerase protein samples in SDS-PAGE profile.
Loading
Band No.
Error
Rf (µ)
material
Error
ExpMW
Error On
Protein
Theo. MW
% error
On
(kDa)
ExpMW
identity
(kDa)
on MW
94.05 [1]
15.3
Lysozyme
14.3 [2]
11.3
Taq protein
94.05 [1]
13.9
Lysozyme
14.3 [2]
14.0
94.05 [1]
13.0
LogMW
on Rf
LogMW
(kDa)
2021 Taq,
1
0.156
0.015
2.084
0.071
121.46
0.16
25x
2
0.191
0.010
2.035
0.072
108.42
0.17
Taq protein
(Lane 1)
3
0.229
0.010
1.982
0.073
96.03
0.17
Taq fragemnt
4
0.480
0.025
1.627
0.090
42.36
0.21
5
0.675
0.025
1.352
0.107
22.46
0.25
6
0.781
0.010
1.202
0.117
15.92
0.27
Thur Taq,
1
0.161
0.010
2.078
0.075
119.58
0.17
5x
2
0.195
0.015
2.030
0.074
107.16
0.17
(Lane 2)
3
0.233
0.010
1.976
0.074
94.54
0.17
4
0.261
0.010
1.936
0.075
86.40
0.17
Taq fragemnt
5
0.277
0.010
1.914
0.076
82.11
0.17
Taq fragemnt
6
0.374
0.010
1.777
0.082
59.79
0.19
Taq fragemnt
7
0.479
0.030
1.629
0.098
42.53
0.23
8
0.671
0.030
1.357
0.114
22.73
0.26
9
0.774
0.010
1.212
0.116
16.29
0.27
10
0.981
0.030
0.920
0.143
8.31
0.33
2022 Taq,
1
0.197
0.010
2.027
0.072
106.32
0.17
Taq protein
10x
2
0.280
0.010
1.909
0.076
81.15
0.18
Taq fragemnt
(Lane 3)
3
0.676
0.010
1.350
0.107
22.38
0.25
4
0.781
0.020
1.202
0.120
15.92
0.28
Lysozyme
14.3 [2]
11.3
Wed Taq,
1
0.203
0.010
2.018
0.072
104.26
0.17
Taq protein
94.05 [1]
10.9
5x
2
0.284
0.010
1.904
0.076
80.20
0.18
Taq fragemnt
(Lane 4)
3
0.676
0.020
1.350
0.110
22.38
0.25
4
0.782
0.020
1.200
0.120
15.85
0.28
Lysozyme
14.3 [2]
10.9
Standard
1
0.101
0.010
2.163
0.069
145.44
0.16
Taq
2
0.202
0.010
2.020
0.072
104.67
0.17
Taq protein
94.05 [1]
11.3
(Lane 6)
3
0.306
0.030
1.874
0.087
74.74
0.20
Taq fragemnt
4
0.341
0.010
1.824
0.080
66.72
0.18
Taq fragemnt
Note: The experimental Taq polymerase samples were extracted from E. coli BL21(DE3). The proteins
sample were ran on 10% SDS gel, and operated for 23 minutes under 250V. The ladder protein used
was Precision Plus Protein Unstained Standards - Cat. # 161-0363). The detailed information for all
bands are calculated and are shown in the Excel document attached.
All the results were calculated by EXCEL, and the data used was retrieved from
ImageLab software. The theoretical molecular weight of Taq polymerase (832 amino
acids) was reported as 94.05 kDa [1]; the theoretical molecular weight of lysozyme
was reported as 14.3 kDa [2].
Figure 4. Western Blot profile of all experimental Taq polymerase protein samples extracted
from E. coli BL21(DE3). Primary antibody used was Anti-Taq Monoclonal Antibody (8C1) (eEnzyme:
MA-029-0250), and the secondary antibody used was Peroxidase-conjugated AffiniPure Goat
Anti-Mouse IgG (H+L) (Jackson ImmunoResearch Laboratory Inc.: 115-035-003). Trans-Blot Turbo
Transfer System (BIO-RAD Inc.) was used to transfer proteins to the membrane. The BioRad
ChemiDoc XRS+ system with ImageLab software were used to image and visualize the gel.
The three sole bands in each lane manifested the identity of Taq polymerase used in
this experiment. The antibody used had a very high specificity to the Taq polymerase
and the sensitivity was also strong when all three bands had a high signal intensity.
Sample calculation
 2021 Taq, 25x (Lane 1), Band 2:
Standard curve: y= -1.4132x+2.3055
① Error of slope: 0.12198
② Error of intercept: 0.06644
Rf = 0.191
Error on Rf:
0.20−0.18
2
= 0.010
Log(MW): Log(0.191) = 2.035 kDa
Error on Log(MW):
2

2
 0.12198   0.010  2
2
(-1.4132 0.191 
 
 )  0.06644  0.072(kDa)

1
.
4132
0
.
191

 

Exponential MW:
10 2.035  108.42(kDa)
Error on ExpMW:
2.303  0.072  0.17(kDa)

Theoretical MW of Taq polymerase [1]: 94.05 kDa
% error on molecular weight:
(108.42  94.05)kDa
100%  15.3%
94.05kDa
3. RT-PCR
Figure 5. Amplification curves in semi-logarithmic view obtained from serial dilutions of a DNA
template(2.86 kbp). Black line: Threshold of amplification of a positive sample. The values
obtained were retrieved during the amplification of each dilution. The dye used for quantifying the
DNA content was EvaGreen Dye (Biotium: 31000).
30
25
CT value
20
15
10
y = -3.9477x + 7.6617
R2 = 0.9953
5
0
-6
-5
-4
-3
-2
-1
0
Log(starting quantity)
Figure 6. Standard curve of CT value versus Log(starting quantity) of serial dilution DNA
samples. The values obtained were retrieved during the amplification of each dilution. The dye used
for quantifying the DNA content was EvaGreen Dye (Biotium: 31000). By plotting this curve and
using EXCEL, the standard curve was alculated to be y= -3.9477x+7.6617, R2=0.9953. The LINEST
function was provided in EXCEL file.
Figure 7. The melt curve regarding the DNA template PCR product. The melting temperature of
the DNA PCR product was detected to be 82.3 oC.
Table 5: RT-PCR analysis during the quantification cycle. The dye used for quantifying the DNA
content was EvaGreen Dye (Biotium: 31000). The Bio Rad CFX Connect Real-Time PCR detection
system was used to detect the content of DNA during amplification reaction.
Standard
Starting
Log(starting
DNA
quantity
quantity)
template
(ng)
(ng)
Cq
Average Cq
STD Average
Cq
1. 11.4
Std-01
0.1
-1
2. 12.03
11.81
0.35
15.59
0.39
18.82
0.51
23.94
0.08
27.37
0.31
3. 11.99
1. 16.04
Std-02
0.01
-2
2. 15.38
3. 15.34
1. 19.41
Std-03
0.001
-3
2. 18.55
3. 18.51
1. 24.03
Std-04
0.0001
-4
2. 23.91
3. 23.87
1. 27.01
Std-05
0.00001
-5
2. 27.57
3. 27.53
 Calculation of efficiency of RT-PCR and % Efficiency:
1. Efficiency of RT-PCR: 101( 3.9476)  10 4.9476  1.792
2. % Efficiency: (1.79  1) 100%  79.2%
4. PCR
Figure 6. PCR analysis of a test DNA sample (2.84 kbp). The sample was ran on 1% agarose gel for
at 150V for about 20 min, staining with Gel Red dye. A 10 kb ladder (Bio Basic Inc. Cat. No M101R-1)
was used. The BioRad ChemiDoc XRS+ system with ImageLab software were used to image and
visualize the gel.
1,2
1
0,8
Log(kb)
0,6
y = -1.7598x + 1.2572
R2 = 0.9942
0,4
0,2
0
0
0,1
0,2
0,3
0,4
0,5
0,6
0,7
0,8
0,9
1
-0,2
-0,4
Relative Front
Figure 7. Standard curve of PCR marker shown in figure 4, represented by Log(kb) versus
Relative front. The LINEST function and the R2 is calculated by and represented in attached EXCEL
file.
The standard curve of PCR marker represented by relationship between Log(#kbp)
versus electrophoretic mobility (Rf), was calculated by EXCEL as
y= -1.7598x+1.2572, R2=0.9942. In which, the error on slope was calculated by
EXCEL as 0.05099 and the error on intercept was 0.02497. The DNA template used
here had a theoretical weight of 2.86 kbp. The experimental agarose gel
electrophoresis analysis indicated that the PCR product had a weight of 3.15 kbp,
which was 10.3% different to the theoretical one.

Sample calculation

DNA Template (Lane 2), Band 1:
Rf = 0.431
Error on Rf:
0.45−0.41
2
= 0.020
Log(MW): Log(0.431) = 0.499 kbp
Error on Log(MW):
2

2
 0.05099   0.020  2
2
(-1.5798 0.431 
 
 )  0.02497  0.048(kDa)
  1.5798   0.431 
Exponential # of kbp:
100.499  3.15(kbp)
Error on Exp# of kbp:
2.303  0.048  0.11(kbp)
Theoretical value of kbases for template DNA: 2.86 kbp
% error on value of kbases for template DNA:
(3.15  2.86)kbp
100%  10.3%
2.86kbp
Discussion
Taq polymerase protein sequence was constructed into pTTQ18 plasmid (4563
bp), and was expressed in E.coli BL21(DE3) through induction of IPTG. This bacteria
strain was modified version of E.Coli and protease expression genes were removed,
preventing protein degradation during the expression process. In addition, it carried
the heterogenous gene of T7 RNA polymerase, which was regulated by lacUV5
operator [15]. Therefore, it could express vector containing a T7 promoter. The
pTTQ18 plasmid used was containing a T7 promoter. The Isopropyl
β-D-1-thiogalactopyranoside (IPTG) was used as a galactose analog to activate the lac
operon, as well as the expression of the Taq polymerase enzyme. Since IPTG would
not be degraded, it was used to over-expressing protein in this case, without adding
extra sugar to the cultures.
The Taq polymerase enzyme inserted into the expression vector had a theoretical
molecular weight of 94.05 kDa (832 amino acids) [1]. Since it was highly
thermostable, as mentioned in introduction section, an incubation for 1 hour at 80 oC
was operated to the extracted native Taq polymerase from total proteins, by
denaturing and removing all others unwanted proteins. Therefore, a pure protein
samples acquired in this lab would have a molecular weight closed to 94.05 kDa. For
the purifier enzyme efficiency, no relevant literature values were available, but it
would be closed to 100%. Also, for the SDS-PAGE gel image, there should be only 1
band of Taq polymerase, as well as the Western Blot image.
In terms of the results, as shown in table 4 and in figure 3, the molecular weight
of experimental Taq polymerase was calculated to be 106.32 ± 0.17 kDa, which had a
13.0 % error compared with theoretical value of 94.05 kDa [1]. However, by
vertically comparing to other Taq samples from other sections and even the standard
Taq polymerase sample, the experimental MW for our experimental Taq polymerase
sample made sense. In which, all the MW values obtained were bigger than the
literature one. Here, SDS played a vital role. The movement of proteins were
depended on the occurrence of SDS reagent, which denatured the proteins, making all
the proteins to turn into a form of linearized structure. It also turned all the native
charge of the proteins into negative, to enable proteins’ movement by giving an
current. For these reasons, the uncertainties of protein MW could be fristly attributed
to that the samples protein did not denature completely to form a linearized form. It
could also be due to a high content of basic amino acids. The most possible reason
could be that the samples protein was not being reduced well, leading to a smaller
movement and a higher calculated molecular weight. To further optimize this,
different manufacture’s SDS reagent can be tried. The concentration of SDS reagent
added could be optimized further. Also, a lower voltage and a longer time of running
gel could potentially relieve this issue. In addition, a lower concentration of SDS gel
could also relieve this problem.
In terms of the SDS-PAGE results shown in figure 3 and table 4, the purified 10x
diluted Taq polymerase sample appeared 4 bands in lane 3. Here, the band 1 was
represented the Taq polymerase enzyme, where the molecular weight was indicated
above. The band 4 in this lane 3 was identified to be the lysozyme, which had a
calculated molecular weight of 15.92 ± 0.27 kDa and this value had a 11.3%
difference to its literature one, 14.3 kDa [2]. The band 3 could be a broken Taq
enzyme fragment, since this band appeared in all four experimental Taq samples, and
the signal intensity of this band were consistent with the Taq sample band in lane 1 to
4. The band 2 of the lane 3 could be protein from E.Coli that was relatively stable
under heat condition because it did not appear in standard Taq enzyme lane and it did
not appear in all four experimental Taq samples. All other bands other than these were
impurities of proteins that could be came from the bacteria culture. Regarding to
standard Taq enzyme sample, however, the band 3 and 4 of the lane 6 had a similar
molecular weight to the Klentaq enzyme, which has been reported a MW of 62.4 kDa
[16], where the band 3 had a MW of 74.74 ± 0.20 kDa and band 4 had a MW of 66.72
± 0.18 kDa. Also for the band 1 of the lane 6, it could be due to the mistake made by
the manufacture or contamination occurred during the process of sample storage.
To sum up, among all these experimental samples in figure 3, the lane 3 and lane
4 seemed to have the highest purity of Taq polymerase sample because they had the
least appeared bands. The Thursday Taq sample seemed to have the most impurities,
when there were totally 10 bands appeared in lane 2. However, it seemed to have a
highest content of Taq enzyme acquired, since the band 2 of the lane 2 seemed to have
a much higher signal intensity. To improve the purity of the Taq sample extracted,
longer period of boiling water bath of the samples could be applied. Also, different
bacteria cells lysis technique could be used such as ultrasonic method. To significantly
improve the impurity of the extract enzyme, an affinity tag such as His-Tag could be
used to optimize this protocol. To improve the yield, a higher concentration or higher
amount of lysozyme could be used.
In terms of the Western Blot results shown in figure 4, only one specific band
appeared, after adding specific primary antibody to the experimental sample and
visualizing by adding secondary antibody. This indicated the identity of the Taq
enzyme. The Western Blot image showed a good quality for all samples. Only some
uneven spots were appeared on the blot. To better fix this, firstly was required to
avoid bubbles when transferring the samples. Secondly, to shake the solution during
blocking process might be helpful. Also, to wash the membrane for more times. In
addition, to centrifuge and filter the secondary antibody could also be helpful [17].
Other than Western Blot, the experimental agarose gel electrophoresis analysis
regarding PCR product using a template DNA, was used to identify the Taq enzyme.
As shown in figure 6, it only had a 1 specific band of 3.15 kbp, which was 10.3%
different to the theoretical one, 2.86 kbp. This result further validated the activity and
the identity of the collected sample. Therefore, the Taq polymerase enzyme was
successfully isolated.
The 2022 Taq enzyme sample was more pure than the 2021 one excepting the
Thursday’s sample. The improvement could be attributed to a better protein extraction
protocol and experimental operation. Excepting the 2022 Thursday’s sample, all the
Taq enzyme samples were desalted well because there were no water spots appeared
on the gel. In the case of content, the 2021 Taq enzyme sample seemed like to have a
higher yield based on the signal intensity. As mentioned above, the less purity of 2021
Taq sample could be attributed to a short period of boiling water bath, leading to the
existence of proteins that were not completely denatured in the sample.
The efficiency of the experimental Taq polymerase was detected to be 79.2%
by using RT-PCR. The amplification efficiency was calculated from the standard
curve of CT value versus Log(starting quantity) of serial dilution DNA samples. The %
efficiency was calculated by (Efficiency - 1)×100%. As the melt curve shown in
figure 7, the melt temperature for the product was detected to be about 85 oC, which
was larger than the expected value of 80 oC. This indicated that there was no or only
trace amount of primer-dimer and non-specific byproducts were produced during the
reactions.
In conclusion, this experiment was successful. The Taq polymerase was
expressed and extracted from E.Coli cultures. The SDS-PAGE results validated the
purity and provided an experimental MW of the Taq enzyme, which were closed to
the literature value. The Western Blot and PCR further demonstrated the identity of
the protein obtained. Finally, the RT-PCR and PCR both obtained desired product,
which significantly manifested the functionality of the Taq polymerase acquired.
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