Protein Separation Using Magnetic Nanoparticles

Anthony Maldonado Castro, Félix Vallés

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. Abs – blank

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

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