Expression Purification and Characterization of KsgA Protein

Deleah Pettie
Lab Partner : Michael Polzin
Lab Section A
11/19/2014

Abstract
This study was performed in order to show
how a desired protein of interest can be
obtained by starting with the gene of
interest then using techniques of genetic
modification and cloning in order to amplify
the desired gene then express the protein
from the new plasmid and purify the sample
in order to obtain the desired product. The
objective of the study was to see if the
cloning expression and purifications
techniques would be useful in obtaining the
KsgA protein from a vector. The results
from the study show the KsgA protein was
present in the PCR samples so the
amplification of the gene was successfully
shown by the presence of a bright band in
the agarose gel. The over expression of the
KsgA protein was seen in the time induction
gel samples the band for each time induction
became darker showing a higher presence of
the protein. KsgA was detected in many
samples of the Lysate of cells and in two
fractions of the protein purification process
this was confirmed with an immunoblot
colorimetric blot and SDS-PAGE to show
the purity of the product. Based on the
results the various isolation, expression, and
purification techniques were successful in
producing the KsgA protein.
Introduction
Many advances have led to the development
of techniques that can be used to express
proteins at higher levels and improving yield
also other techniques have been developed
in order to genetically modify proteins so
the purification process is simpler. These
procedures are demonstrated in this study of
protein expression purification and
characterization the protein of interest KsgA
has been modified with 6-His tags in order

to help the purification process with Metal

Chelate Affinity Chromatography Nickel is
used in the column which causes an
interaction with the His-tag allowing for
better purification of the protein. The Histags are also useful in helping detect the
protein of interest through the use of
antibodies. IPTG is used in order to help the
overexpression of the KsgA protein through
time induction. All of these procedures are
performed in order to see how efficient it is
to genetically modify genes in order to
obtain desired product from cloning and
purification.
Materials and Method
Colony PCR
Five polymerase Chain Reaction
amplification reactions were prepared for
colonies plated by the teaching assistants
each plate labeled A,B,C and D and the fifth
reaction has no colony and is the negative
control. For the five reactions a master mix
was prepared 125ul total for the five
reactions containing 12.5 ul of PCR 10X
Buffer 7.5 ul of MgCl2 25mM stock
concentration, 5ul dNTPs 5mM stock, 5ul
for primers forward and reverse, 82.5 ul of
sterile water and 1.5 ul of Taq added to the
five reactions individually equaling 7.5 ul
total. 23.5 ul of the PCR master mix was
placed in the respectively labeled tubes for
the colonies A, B, C, D and negative control
a colony was picked using a sterile pipette
from each plate and placed in the PCR
reaction tube. Taq was added last to the
reactions and the PCR program was run. The
set up for the PCR cycles were 94 Celsius
for 4 min followed by 25 cycles of 94C for
30 seconds then 55C for 1 minute then
72C for 1 minute and the PCR program
ended with a cycle at 72C for 10 min. Once

PCR was complete a 0.8% agarose midi gel

in 1X TAE buffer with 25% SyberSafe
added was cast. 15 ul of each PCR reaction
put into a new tube and mixed with 3 ul of
6X sample loading buffer the 18ul samples
were loaded into the wells 1-5 of the gel
with 10 ul of 1 Kb marker in lane 6. The gel
power supply was run at 90 volts for 30
minutes once the run was completed the gel
was imaged using a photo-doc system. The
results of the gel were used in order to find
the right culture to be used.
Isolation, Quantification, Digestion, and
Transformation of DNA
Four cultures 10 ml aliquots were obtained
and 1.5 ml of culture was transferred to
micro centrifuge tubes properly labeled and
were all spun down for one minute at 8,000
rpm and supernatant was discarded. This
step was repeated a second time and then the
pellet was suspended in 250 ul of P1 buffer
from QIAprep the re-suspended pellets were
transferred to clean microcentrifuge tubes.
250 ul of P2 buffer was added to each tube
and gently inverted 4-6 times 350 ul of
Buffer N3 was added to the tubes and then
they were inverted 4-6 times. The tubes
were then centrifuged for 10 minutes at
13,000 rpm allowing a white pellet to form.
Supernatants were applied to a QIAprep spin
Column then centrifuged for 60 seconds at
13,000 rpm. Columns were washed by
adding .75ml Buffer PE and centrifuged for
30-60 sec flow through was discarded. The
samples were centrifuged again for 1 minute
to remove residual wash buffer then the
QIAprep column was placed in a clean 1.5
ml microcentrifuge tube to elute the DNA
with 30ul of sterile water followed by
centrifugation for 1 minute. The DNA
samples were quantified using the
spectrophotometric method of the nanodrop
and the absorbance was read at 260nm for a
2ul sample. Once the purified plasmid was

obtain four different reactions were setup for

restriction enzyme digestion of the DNA
with enzyme XBA substitute for SmaI and
HpaI 1 ug of DNA was placed into four
different sterile microcentrifuge tubes the
first tube had 1 ul of both enzymes XBA and
HpaI the second tube had contained only
XBA, the third tube had only HpaI and the
fourth tube had no restriction enzyme for the
negative control. Once enzyme was added
2ul of buffer J was added to all the reactions
and the remaining volumes were of sterile
water in order to make the total volume of
the reaction 20ul the mixtures were
incubated overnight. After the digestion was
complete the digested product was analyzed
using agarose electrophoresis. For the
preparation of competent cells 10ml aliquots
of the inoculated LB broth with BL21 phage
resistant bacteria was spun at 4C for 10
minutes at 5,000 rpm once pellet was
formed supernatant was decanted and the
pellet was re-suspended in 5ml of ice cold
100mM CaCl2 the sample was then placed
on an ice bath for 20 minutes then
centrifuged again at 4C for 10 minutes at
5,000 rpm and the supernatant was decanted.
The pellet was then re-suspended in 0.9ml of
ice cold 100mM CaCl2 from the cells 200ul
would be used for each transformation
reaction. 100 ng of plasmid DNA pET15bKsgA-his was added to 200ul of competent
cells BL21 as a negative control 100ng of
plasmid DNA pUC19 was added to 200ul of
competent cells both mixtures were
incubated on ice for 20 minutes then heat
shocked t 42C for 90 seconds. After the
heat shock the reactions were placed on ice
for 2 minutes then with sterile technique 0.9
ml of LB media was added to the reaction
tubes then the reactions were placed on a
shaker for 1 hour at 37C. 100 ul of positive
control which was the TA KsgA and 50 ul of
the TA KsgA were plated on respectively
labeled plates and one plate contained 100 ul
of the experimental KsgA and the last plate

had 100ul of the negative control. All the

plates were incubated for 12-16 hours at
37C.
Over Expression of KsgA-His Tagged
Protein in BL21
Two 10ml aliquots of sterile LB had a final
concentration of 80ug/ml ampicillin added
to them one was labeled for BL21with pET
15b-KsgA-his and the pUC19 as a control
both samples were inoculated with the
proper vectors then incubated at 37C
overnight on a shaker. Ampicillin was added
to two new 250ml flasks containing 125ml
of LB media the ampicillin was added to
final concentration of 80 ug/ml. 3ml was
taken from each overnight culture and
inoculated into the respectively labeled flask
BL21 with vector pET15b-KsgA-his and
pUC19 as a control. The cultures were
incubated in a shaker for 3 hours at 37C.
The time induction procedure was
immediately started after the incubation was
complete time point zero t=0 1.5ml from
each culture was set aside for SDS-PAGE
analysis then 1mM of IPTG was added to
both of the cultures then put into the
incubator during the incubation the reaction
was being timed for time point t=0.5 which
was 30 minutes and time point t=1 which
was 1 hour at each time point two samples
were collected a 1.5ml aliquot for protein
gel and a 50ml aliquot for purification all
samples were kept on ice. To increase
protein concentration of 1.5ml aliquots the
samples were centrifuged at 5,000 rpm the
supernatant was removed then the pellets
were re-suspended in 200ul of sterile water.
In the cold room the 50ml aliquots were
centrifuged at max speed for 10 minutes to
pellet the cells then the media was discarded
and the pellets were saved for later. A SDSPAGE gel was performed on the time
induction samples for the t=0 t=0.5 and t=1
hour for each sample 15 ul of protein was
mixed with 1 ul of DNAse and 10ul of 4X
LDS sample loading buffer. The samples

were run in A 10% Bis-Tris pre-cast gel. In

lanes 1 and 2 of the gel 25 ul of pUC19 t=0
and t=1 hour were added in lanes 3,4 and 5
25 ul each of the t=0 t= 0.5 hour and t=1
hour for the BL21 pET15b-KsgA-his and
lane 6 contained 8-10ul of marker see Blue
2+. The gel was run at 180 volts 100mA for
approximately 45-50 minutes the gel was
rinsed three times in distilled water then
placed in 50ml of Simply Blue overnight in
a plastic tray on a shaker. Once the
incubation of the gel was complete the gel
was rinsed and imaged.
Purification and Protein Analysis of Histagged KsgA
The time induction pellet with the best
expression of the KsgA protein seen from
the SDS-PAGE gel was thawed and had 5ml
of Lysis buffer added to it 0.2 ml of the
sample was saved for protein determination
and labeled starting cell pellet. The sample
was then sonicated for 45 seconds on and 3
minutes off for 5 cycles. After sonication the
samples were centrifuged at maximum
speed for 15 minutes using a clinical
centrifuge in the cold room and 0.5ml
aliquot of supernatant was saved and labeled
Lysate for protein determination and SDSPage analysis. The supernatant was decanted
into a 12ml tube containing 1 ml Ni-NTA
beads and the sample was placed on a rocker
in the cold room for 30-45 minutes to allow
binding. 300 ul of lysis buffer was added to
the column and the volume was used to start
the flow and pre wet the column. The lysate
and Ni-NTA beads were added to the
column and the volume went through the
column twice and the flow through was
collected and labeled FT and saved for
protein determination and SDS-PAGE
analysis. The column was then washed with
10ml of wash buffer and the wash fraction
was collected and labeled wash. The protein
was then eluted using 5ml of elution buffer 5
microfuge tubes were labeled F1-F5 for 1ml
of elution in each tube. A Bradford assay

was performed on all fractions with 100ul of

sample and 0.5 ml of Bradford reagent the
two samples with the most intensity of blue
were chosen for dialysis in the cold room a
1L beaker of cold dialysis buffer was
prepared the fractions with the most protein
were then placed in the dialysis tubing and
placed in the buffer overnight with the
buffer being replaced once after the first 4
hours. A Bradford assay was performed on
the protein with Bovine Serum Albumin as
the standard then the results were read by a
Cary 50 and estimated by a Nanodrop. Two
SDS-PAGE analysis gels were ran one for
protein determination the second for the
western blot for sample preparation the
volumes were doubled 16-20 ul of Standard
See Blue +2 was added 30 ul of lysate was
used and 10 ul of 4X LDS sample buffer and
40 ul of the two purified protein fractions
with 10ul of 4X LDS sample buffer and 5 ug
of KsgA purified protein provided by the
teaching assistants. Lane 1 of the gel
contained See Blue 2+ marker lane 2 had the
lysate lane 3 had fraction with highest
protein concentration lane 4 had the fraction
with the 2nd highest protein concentration
lane 5 contained purified KsgA and lane 6
was empty. The iBlot transfer system was
used to blot and transfer the gel membrane
once the transfer was complete the layer was
stained with Simply Blue. The membrane
was placed in a tray with 10ml of
BSA/TBST for 1 hour at room temperature
with shaking then the membrane was
washed twice with 15ml of TBST for 10
minutes then the blot was incubated for 1
hour with shaking in 10 ml of HisProbeHRP working solution. Then the membrane
was washed four times with 15 ml TBST for
10 minutes then the blot was incubated with
7.5ml of SuperSignal West Pico Substrate
working solution for 5 minutes the blot was
then placed in Saran wrap and had bubbles
and excess liquid removed once this was
complete the blot was imaged.

Results
The results for the colony PCR and the
restriction enzyme digestion can be seen in
Figure 1. On the agarose electrophoresis
some of the sample bands are easy to detect
and image while others are difficult to see.
The PCR results show the pUC19 vector
band and the TA KsgA bands the positive
control for PCR is also visible in the figure.
The negative control is not present in the
image so this is verification of what no PCR
activity would look like on the gel.
The restriction enzyme digestion indicates
the fragments created from the plasmid and
can be used to determine a plasmid map for
the vector specified. In the lane indicating
no enzyme digestion there should be one
band to indicate how large the vector should
be without any restriction sites cut. XBA and
HpaI lane should show multiple bands and
be compared to the markers in order to
figure out each size of the fragment while
the lanes with only XBA and HpaI should
only have one band to show the plasmid
would be cut but it would still be a long
uniform strip.
The Time induction gel results can be seen
in Figure 2.each sample and the time points
are properly labeled in the image and the
markers where the KsgA protein is between
are indicated in the image as well. The
bands in each of the time induction samples
for the BL21 pET15b-KsgA+His
determined to be the KsgA protein are
marked in the image as well these results
were used to determine which time
induction sample expressed the KsgA
protein the most.
The Results for the protein analysis of KsgA
can be found in Figure 3. Figure 4. Figure 5.
and Table 1.. Table 1. shows the results from
the Bradford assay protein determination of
the KsgA samples from the starting cell
pellet the lysate the flow through the wash
and fractions 2 and 3 all of these samples
were taken from the protein purification

process the data shows the absorbance of

each sample at 595nm and this absorbance
was used in a standard curve from previous
experiments to determine the concentration
of protein in ug/ml these results can be used
to determine how purified the protein
samples are for KsgA.
Figure 4. is an image of A SDS-Page gel in
order to analyze the various proteins present
in the samples the more bands seen on the
gel the more contaminated the protein
sample is. Figure 3. Is an image of the
immunoblot for the protein samples and
Figure 5. is an image of a colorimetric blot
both of these techniques were used to detect
the presence of the KsgA protein.
Discussion
For this experiment there was a lot of error
that had occurred for the first part of the
experiment the first PCR reaction failed due
to the KsgA containing colonies not
expressing the KsgA gene the first gel had
no indication of any presence of the gene for
KsgA so a second agarose electrophoresis
was performed with PCR products and
restriction digestion products shown in
Figure 1. Unfortunately this gel was not well
processed or imaged because all of the
bands are large blurs and so the ladder
markers are hard to indicate also there are
many dark spots on the image for unknown
reasons which could possibly hide bands
that may have been otherwise detected.
Because the bands cannot be detected
accurately there is a great amount of error in
the interpretation of the restriction enzyme
digestions and the fragments created. The
estimated restriction enzyme map is based
on the pET15b vector map that has XBA and
HpaI as restriction sites the estimated map
can be seen in Figure 6. Because the
information from the agarose gel is hard to
interpret the plasmid map could not be
accurately created. Based on the information
from other vector maps of pET15b if the
plasmid was digested with Both XBA and

HpaI there would be two fragment pieces if

only one of the enzymes was used there
would be one band on the gel indicating the
plasmid would be cut but would remain one
long piece of DNA. The no enzyme sample
would be one band with a high bp count to
show the size of the plasmid. In the image
the lane can be seen but the band is not
present. In the image can be seen attempts to
define the markers and bands of the samples
but the lighting of the bands blended within
one another it is difficult to distinguish
where one band begins and ends.
The results from the Time induction were
quite successful the gel was imaged and the
See Blue +2 marker was very distinct the
KsgA protein is known to be about 32 kDa
so the protein band would be between the 28
and 38 kDa bands which are indicated in the
image also the band had to be one present in
the BL21pET15b-KsgA-His vector samples
but not seen in the pUC19 time induction
samples this would verify the indicated
bands as the KsgA protein. For the protein
purification the sample with the best
expressed KsgA would need to be used so
the time induction 1 hour sample had the
darkest band so it was chosen for
purification. Based on the results from the
protein determination shown in Table 1. the
lysate was indicated to have the highest
concentration of protein this is possibly due
to high contamination of various proteins
not necessarily the desired KsgA protein.
Something peculiar about the data is the big
concentration differences between fraction 2
and 3 of the protein purification this could
possibly be due to contamination of the
sample because it is difficult to keep track of
the different sample volumes flowing
through the column and making sure all of
the original sample is within the fractions.
The western blot colorimetric blot and SDSPAGE of the protein purification samples
were useful methods in detecting whether
the desired KsgA protein was present.

Figure 4. the SDS-PAGE analysis of the

samples show that the KsgA protein is
present in fraction 2, fraction 3, and the
lysate this is shown by the TA KsgA which
is of high purity this is confirmed by the
presence of only two strongly visible bands.
Fraction 2 and 3 have the KsgA protein and
have many other bands this indicates there
are a lot of other undesired proteins that had
been expressed the lysate also indicates the
presence of KsgA and has many other bands
showing other proteins. Fraction 2 had a
presence of many more bands than fraction
3 this would confirm why the protein
determination had such a difference
concentration. Figure 3. and Figure 5. are
the results from the immunoblot and the
colorimetric blot the results from the
colorimetric blot are difficult to see but a
slight mark can be seen in the image where
the KsgA band should be present the TA
KsgA cannot be detected this could be due
to protein degradation. The immunoblot is
very distinct showing dark bands where the

KsgA is present these results verify the

presence of the desired protein because the
antibodies only stick to specific protein
targets. For future experiments would like
to have better PCR and restriction enzyme
digestion results in order to more accurately
determine the plasmid map for the vector.
Also would be interesting to use various
purification methods such as affinity
chromatography or size exclusion
chromatography in order to better purify the
protein so there is not as much
contamination from other proteins.

Figures and Tables Legend

Figure 1. Image of Agarose electrophoresis for PCR reactions and restriction enzyme digestion
of pET15b vector with KsgA insert lanes 1-5 are the restriction digestions with the respective
enzyme added as the label for the lane in lane 1 XBA and HpaI was added in lane 2 only XBA

was added lane 3 only HpaI lane 4 had no enzyme lane 5 was the 1kb marker and lanes 6-10 are
the PCR reaction samples each lane is labeled with the respective PCR sample TA KsgA pUC19
positive control negative control and 1kb marker in the last lane.

Figure 2. image of SDS-Page for analysis of time induction products for pUC19 t=0 lane 1, lane
2 pUC19 t=1 hour, lane 3 BL21+pET15b-KsgA-His t=0 lane 4 t=0.5 hour lane 5 t=1 hour, lane 6
See Blue +2 marker labeled bands that indicate 38kDa and 28kDa and marked the bands that are
KsgA protein.

Figure 3. Immunoblot of His-tagged KsgA protein fractions 3 and 2 and Lysate from purification
of protein the dark spots are where the antibodies are present indicating the desired protein KsgA
is present in the fractions and the lysate.

Figure 4. SDS-PAGE of KsgA-His-tagged Protein fractions and lysate with See Blue marker and
a sample of TA KsgA Lane 1 See Blue Marker Lane 2 Lysate KsgA Lane 3 Fraction 2 of protein
and lane 4 fraction 3 of protein and the last lane contained TA prepared KsgA protein.

Figure 5. Colormetric blot of KsgA fractions 3 and 2 with lysate sample and TA KsgA the band
for KsgA is circled and labeled the TA KsgA is labeled in the lane it was present the next lane

was fraction 3 for the KsgA-His-tagged protein and the next lane was fraction 2 then lysate
sample followed by See Blue marker.

Figure 6. estimated restriction digestion map for the pET15b+His+KsgA the entire plasmid is
about 5708 bp this information is found from other restriction maps of pET15b and the
restriction sites of XBA and HpaI are given in bp the length of the digested fragment is
calculated and the remaining plasmid without the XBA and HpaI fragment the actual numbers
are an estimation of the plasmid map.
Table 1. Bradford assay results for the absorbance and protein concentration in the various
protein purification samples starting cell pellet, lysate, flow through, wash, fraction 2, and
fraction 3 for the KsgA+His protein Absorbance was read at 595nm and concentration is in
ug/ml.
sample
starting
cell pellet
lysate
flow
through
wash
fraction 2
fraction 3

abs
595nm
0.865

conc.
(ug/ml)
752.0

1.4054
0.9825

1243.3
858.8

0.1892
0.7051
0.1929

137.6
606.6
141.0

References
1.
2.
3.
4.

Robyt F. J., White J. B.,(1987) Biochemical Techniques Theory and Practice

Zabotina O., Dispirito A.(2014) Techniques in Biochemical Research Laboratory Manual
Novagen(2014) pET-15b Vector pET-15b Restriction sites
New England Biolabs (2014) pUC19 vector