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Protein Separation Using Magnetic Nanoparticles
Anthony Maldonado Castro, Félix Vallés Feliciano
Tissue plasminogen activator (tPA), an enzyme found in endothelial cells, catalyze
plasminogen to plasmin, which is an enzyme responsible for blood clot breakdown. tPA
can be obtained from pig hearts and ovaries, human postmortem vascular perfusates,
uterine tissue, and postexercise blood. In this experiment it will be extracted from
mammalian cell culture broth (HeLa cells).The purpose of this investigation is to isolate
tPA from HeLa cells so that it can be applied to diverse blood disorders. Within these
applications, thrombolytic therapeutics, malignant tumor treatment, reducing the risk of
hemorrhagic transformation, and many more could be mentioned. Magnetic nanoparticles
(MNPs) will be used as a separating medium, given that they are more effective than other
traditional separating methods since they are fast, scalable, easily automated and separated
from other suspended solids. Moreover, they reduce the pretreatment and chromatography
stages into a single step isolation when combined with affinity binding. Fibrin zymography,
a technique used to determine the presence of hydrolytic enzymes, will be used to show
whether or not tPA is active. Sodium dodecyl sulfate polyacrylamide gel electrophoresis
(SDS PAGE), is a technique that determines the presence of proteins and will be used to
tell if tpa were successfully isolated. The tPA will be bound to the MNPs, the spent will be
obtained from the load using MNPs, several washes and elutions will be performed, the
SDS PAGE and zymography will run and finally the absorbance of each sample are read to
prepare a chromatogram. tPA was successfully isolated after performing these steps for the
second time and used silver staining instead of Coomassie Blue staining.
Introduction
Proteins are macromolecules that consist
of up 20 different amino acids. They play
key functions in the living system such as
carrying oxygen, controlling the sugar
levels in the blood and defending against
foreign cells, pathogens and bacteria.
Isolation of proteins may have different
objectives such as catalyst usage,
therapeutics, dietary supplements and
structure studies.
Two types of chromatographic
techniques will be used, sodium dodecyl
sulfate polyacrylamide gel electrophoresis
(SDS PAGE) and fibrin zymography.
SDS PAGE, a technique in which
proteins are separated according to their
size and electrophoretic mobility, will be
used in this project to estimate the
concentration of protein in each sample.
On the other hand, fibrin zymography, a
technique used for the detection of
hydrolytic enzymes, will be used to
determine the enzymatic activity of each
sample. It can be used for peptidase
investigation, identifications and
characterizations in biological living
systems. For example, it could be used to
detect low levels or absence of thrombin,
the active form of prothrombin, which
converts fibrinogen to fibrin, which is
essential for blood coagulation.
Nanoparticles are more efficient than
other traditional separation methods due
to their superior qualities such as higher
surface to volume ratio, efficient
dispersibility and absence of internal
diffusional limitations. Magnetic
nanoparticles (MNPs) will be used in this
experiment since they are fast, scalable,
easily automated and separated from
other suspended solids. Combined with
affinity binding, MNPs reduce the
pretreatment and chromatography stages
into a single step isolation.
Tissue plasminogen activator (tPA), is an
enzyme found in endothelial cells
involved in the breakdown of blood clots
by catalyzing plasminogen to plasmin, an
enzyme responsible for clot breakdown.
“t-PA is a poor plasminogen activator in
the absence of fibrin. However, in the
presence of fibrin, however, its activity is
two orders of magnitude higher. The
kinetic model indicates that both t-PA and
plasminogen bind to fibrin in a sequential
and ordered way, yielding a cyclic ternary
complex in which t-PA has a markedly
enhanced affinity for its substrate
plasminogen.” (Collen D, Lijnen HR.
2009) “Since tPA is free of immune side
effects and has short half-life, it is
considered an excellent thrombolytic
agent for medical use.” (Byong-Gon P, et
al. 2000)
The isolation of this enzyme could be
applied for fibrinolysis, the breakdown of
fibrin clots to prevent thrombus
formation. Another application for this
enzyme is the inhibition of malignant
protease activity secreted by malignant
tumors using plasma α2-antiplasmin, a
plasmin inhibitor that can rapidly
inactivate free plasmin in the blood.
(Collen D, Lijnen HR. 2009). It could
also be used to reduce the risk of
hemorrhagic transformation when
reducing hypertension during fibrinolysis,
since tPA-induced hemorrhage depends
on blood pressure. (Emiri T, et al. 2001)
Our goal is to isolate tPA from
mammalian cell culture broth by binding
MNPs to tPA, preparing the load, which
consist only of HeLa cells, obtaining the
spent by magnetically separating the load
and MNPs solution, performing several
washes, which will remove all the
nonspecific bound proteins, and
performing several elutions, which will
separate tPA from MNPs by lowering pH
and results in pure proteins. The marker,
load, spent and the 3 elutions will then be
incorporated into the SDS PAGE and
fibrin zymography, which will then
indicate their absorbance. Finally, the
absorbance of these samples will be read
to know if proteins and enzyme activity
are present and then prepare a
chromatogram, a pattern formed on the
adsorbent medium by the sheets of
samples separated by chromatography.
Materials and Methods
In a 15mL tube, MNPs were suspended in
4.0mL of PABA binding buffer, and were
sonicated for 5 minutes. Next the buffer
was decanted, using a magnet, and
substituted with 4.0mL of new binding
buffer, then were placed on a rocket at
4ºC for 5 minutes, after which the buffers
was again magnetically decanted. This
process was repeated tree times. Next
1.0mL of HeLa Broth solution was added
to the MNP; 250µL of the HeLa Broth
solution were saved and labeled as
“Load”. Then the tube was placed on on a
rocker at 4ºC and at 150 rpm, for 1 hour.
After the incubation period was over,
using a magnet the solution was decanted
and 1.5mL of the supernatant was saved
as “Spent”. Next the MNPs were washed
using 4.0mL of PABA binding buffer that
was added, and the tube was placed on a
rocker for 5 minutes at 200rpm. After the
time passed 100µL of the wash was
saved. This process was done tree times.
After washing the MNPs to remove non
specific bound proteins, 1.0mL of PABA
Elution buffer was added to the MNPs
and then they were placed on a shaker for
5 minutes at 150rpm. After the 5 minutes,
the supernatant was saved. This process
was done tree times. After this the MNPs
were regenerated using PABA
regeneration buffer, 4.0mL, for 5 minutes
at 200rpm, then decanted. This process
was done tree times, and then the samples
were washed four times with deionized
water for 5 minutes at 200rpm. After this
they were labeled and lypholized for
storing. The absorbance for “Load”,
“Spent”, Washes 1, 2 and 3, and Eluates
1, 2, and tree was measured at 280nm.
Elution buffer was used as blank for the
eluates, and binding buffer was used as
blank for the rest of the samples.
Next the samples were desalted using 3
desalting columns. First the bottoms of
the columns were cut and le to drain, after
which they were filled with water and let
drain, tree times. Next the columns were
filled with 1xPBS and were left to drain.
Now the eluates were dissolved in 2.5mL
of deionized water, then were added to
the columns and let to drain. Next 3.5mL
of 1xPBS buffer was added to the column
and the filtrate was collected in a 15mL
tube labeled “desalted”. After this the
samples were stored at ‐80ºC.
A protein estimation assay was done. For
this 11.5 µL of each sample and 169µL of
BCA were added in a microplate and
incubated at 37ºC for 30 minutes, then the
absorbance was measured. Next an
enzyme activity determination assay was
done. For this to 50µL of the samples,
70µL of Tris/Tween buffer, 30µL of D-
VLK x/10 and Plasminogen x/10 were
mixed and added to a microplate. They
were incubated at 37ºC for 1 hour, and
then the absorbance was measured.
Next an SDS-PAGE was runned using the
samples from the protein estimation, for
analyzing the samples. For this the Load,
Spent and Eluates were mixed with
deionized water and sample buffer, after
which 18µL were added to the wells in
the gel, and it was run at 150 volts until
the samples have run more than halfway
the gel. Also a Fibrin Zymography was
done to measure enzyme activity. For
this the samples were mixed with 10µL of
sample buffer, and then 45µL of each
were loaded to the gel, which was runned
at 4ºC at 125 volts. These procedures
were done to analyze the samples.
Results
After the MNPs based protein separation protocol, the absorbance of the samples war
measured. The following table summarizes the results for this procedure, along with a
chromatogram.
Sample Volume (mL) Abs.280nm Abs – blank 280nm
Blank buffer 1.5 0.047 0
HeLa Load 1.5 1.269 1.192
HeLa Spent 1.0 1.130 1.083
Wash 1 1.0 0.158 0.111
Wash 2 1.0 0.108 0.061
Wash 3 1.0 0.125 0.078
Wash 4 1.0 0.076 0.029
Elution buffer 1.0 0.049 0
Eluate 1 1.0 0.158 0.109
Eluate 2 1.0 0.088 0.039
Eluate 3 1.0 0.083 0.034 Table 1. Absorbance of the samples after the MNPs based separation.
Graph 1. Absorbance of samples in Table 1.
-0.2
0
0.2
0.4
0.6
0.8
1
1.2
1.4
0 2 4 6 8 10 12
Ab
s a
t 2
80
nm
Vulume (mL)
Chromatogram for separation of TPA from HeLa cell culture
using MNPs
Series1
The samples were used for a protein estimation assay and an enzyme activity assay.
Sample Abs405 Abs - Blank Activity Average Act.
Load HeLa 0.149 0.091 0.1075 161.25
Load HeLa d 0.182 0.124
Spent HeLa 0.158 0.1 0.0995 149.25
Spent HeLa d 0.157 0.099
Eluate 1 0.069 0.011 0.0115 17.25
Eluate 1 d 0.07 0.012
Eluate 2 0.056 -0.002
Eluate 2 d 0.055 -0.003
Eluate 3 0.062 0.004
Eluate 3d 0.061 0.003
Table 2. Ensyme activity assay; absorbances and average activity for each sample.
Figure 1. Fibrin Zymography. From left to right: HeLa Load, Spent, Eluate 1, 2 and 3.
Activity can be appreciated only on HeLa Load, Spent and eluate 1.
Sample ABS 562nm Abs - Blank Concentration Conc. Average
Load HeLa 2.31 2.223 4.98654105 4.384253
Load HeLad 1.773 1.686 3.781965007
Spent HeLa 1.946 1.859 4.170031404 3.835801
Spent HeLad 1.648 1.561 3.501570211
Eluate HeLa1 0.109 0.022 0.049349484 0.026918
Eluate HeLa1d 0.089 0.002 0.004486317
Eluate HeLa2 0.095 0.008 0.017945267 0.020188
Eluate HeLa2d 0.097 0.01 0.022431584
Eluate HeLa3 0.095 0.008 0.017945267 0.01794
Eluate HeLa3d 0.673 0.586 1.314490803
Table 3. Protein estimation assay. Absorbance and estimated concentration of TPA in
samples.
Figure 2. SDS-PAGE, from left to right: marker, Load, Spent, Eluates 1, 2 and 3. Only
HeLa spent shows concentratioi. The gel was satained with Coomasie Blue.
Figure 3. Second SDS-PAGE, same order as in Figure 2. Here the Load, Eluate 1 and 2
show concentratrions.
Discussion
After the protein separation with MNPs
the absorbance of each sample was
measured in order to estimate protein
concentration, (tPA). The higher
concentrations were from the HeLa Load
and Spent. As for the eluates, the higest
concentration is for eluate 1. In Table 1,
the absorbance of the samples can be
observed, eluate 1 having an absorbance
of 0.109 at 280nm. In Graph 1, the
chromatogram for the samples in Table 1
is illustrated. As predicted from the
values in the table, the HeLa Load and
Spent have higher absorbance, following
the eluates. The estimated protein
concentrations for the samples are listed
in Table 3, where it can be seen that
elution 3 is the one with the highest
concentration; the average is 0.026918
kDa. This protein estimation assay
suggests that tPA was effectively
separated from the HeLa Broth.
In Table 2, the results for the enzyme
activity assay are listed. According to this
data, only eluate 1 should show enzyme
activity, since is the only eluate with
positive numbers in the data.
After the absorbances were measured and
the concentrations and activities were
estimated, an SDS-PAGE and a Fibrin
Zymography were performed. Figure 1
ilustrates the results for the zymography.
Only the HeLa Load and Spent show
enzyme activity. These are the two rows
at the left of the image, were clear spots
can be observed. These clear spots mean
that the enzyme degraded fibrin, the
substrate used for the zymography, and
therefore the enzyme (tPA) is active. The
elutions, however, show very little or
perhaps no activity, as it can be observed
in the gel, if compared with the HeLa
Load and Spent. An explanation for this
could be that the protein concentrations
were too low or that the enzyme may
have lost activity or could have been
denaturalized.
In Figure 2 the results for the first SDS-
PAGE are illustrated. In this image only
the marker and the HeLa Load have
expressed. Again here the possible
explanations could be that the protein
concentrations were too low. Also this gel
was stained using Coomassie Blue, which
is not very sensitive when concentrations
are low. A second SDS-PAGE was
performed, this time using silver stain,
and testing the same samples as in SDS-
PAGE #1. Silver staining was used
because it is more sensitive when sample
concentrations are low. Figure 3 show
the results for this second trial, were the
HeLa Load, Elution 1 and 2 expresed.
However, these results might have been
due to an experimental error, since the
band in elution 1 is way too dark,
according to the average concentration
listed in Table 3. Therefore, the band in
elution 1 should be “Spent” and the band
in elution 2 should be “elution 1”; this
because elution 1 is the one with the
highest concentration and enzyme
activity.
In conclusion, according to the
experimental data, MNPs are an effective
way for separating proteins; in this case
tPA, from mammalian cells, although in
very low concentrations.
Acknowledgements
Special thanks to:
Vibha Bansal, Ph.d
The RISE Program
Alexandra Rosado
Osvaldo Vega
José J. Rosado
Natalia Espada
Mariana León
Reference
Collen D, Lijnen HR. (2009)
Arteriosclerosis, Thrombosis, and
Vascular Biology. American Heart
Association 29: 1151-1155.
Byong-Gon P, Joo-Mi C, Chang-Jin L,
Gie-Taek C, Ik-Hwan K, et al. (2000)
Development of High Density
Mammalian Cell Culture System for the
Production of Tissue-Type Plasminogen
Activator Biotechnol. Bioprocess Eng.
5(2): 123-129
Emiri T, Yoichi K, Yasuyuki S, Tsuneo
K, Eng HL. (2001) Hemorrhagic
Transformation After Fibrinolysis With
Tissue Plasminogen Activator. American
Heart Association 32: 1336-1340