This document summarizes a study that isolated the protein tissue plasminogen activator (tPA) from mammalian cell culture broth using magnetic nanoparticles. SDS-PAGE and fibrin zymography analyses showed tPA enzymatic activity was successfully separated in the first elution fraction. The results demonstrate magnetic nanoparticles are an efficient method for protein separation that is fast, scalable, and allows single-step isolation by reducing pretreatment stages.

1. PROTEIN SEPARATION
USING MAGNETIC
NANOPARTICLES
Anthony Maldonado Castro
Félix Vallés
Dr. Vibha Bansal
RISE Program

2. Proteins
 Proteins- macromolecules that consist of up to
20 different amino acids
 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
purposes such as catalyst usage,
therapeutics, dietary supplements and
structure studies.

3. What did we use?
SDS PAGE
 Fibrin Zymography

4. Sodium dodecyl sulfate PAGE
 An electrophoretic technique in which proteins
are separated according to size.
 In this particular project it is used to estimate
the concentration of protein in each sample.

5. Fibrin Zymography.
 Fibrin zymography is an electrophoretic
technique for the detection of hydrolytic
enzymes.
 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, that is essential for blood coagulation.

6. Fibrin Zymography vs. SDS
PAGE
 SDS PAGE- used to determine the prescence
of proteins
 Fibrin Zymography- used to determine
enzyme activity

7. Magnetic Nanoparticles (MNPs)
 MNPs are more efficient than traditional
separation methods since they are:
 Fast
 Scalable
 Easily automated and separated from other
suspended solids
 They reduce the pretreatment and
chromatography stages into a single step
isolation when combined with affinity binding.

8. Tissue Plasminogen Activator
(tPA)
 Tissue plasminogen activator (tPA), is a
protease found in endothelial cells involved in
the breakdown of blood clots by catalyzing
plasminogen to plasmin, an enzyme
responsible for fibrin clot breakdown.
 “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)

9. Tissue Plasminogen Activator (tPA)
(cont.)
 “t-PA is a poor plasminogen activator in the
absence of fibrin. However, in the presence of
fibrin, 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)

10. Fibrinolysis- breakdown of fibrin
clot to prevent thrombus
formation
α2-
antiplasmin
tP
A
Plasminoge
n
Plasmin
Fibrin clot
degradatio
n

11. Tissue Plasminogen Activator (tPA)
(cont.)
 “Malignant tumors frequently secrete
plasminogen activator activity, and that their
malignancy correlates with the level of
“malignant protease” secreted. This protease
activity could be inhibited with plasma α2-
antiplasmin, a plasmin inhibitor that can
rapidly inactivate free plasmin in the blood.
(Collen D, Lijnen HR. 2009)

12. Tissue Plasminogen Activator (tPA)
(cont.)
 “This study demonstrates that in this rat
thromboembolic model of stroke, tPA-induced
hemorrhage is dependent on blood pressure
and that pharmacological reduction of
hypertension during fibrinolysis can reduce the
risk of hemorrhagic transformation.” (Emiri T,
et al. 2001)

13. Isolate Plasminogen Activator (PA) from
mammalian cell culture broth.
Objectives:

14. Plasminogen Activator (PA) will be isolated
from mammalian cell culture broth.
Hypothesis:

15. Procedure
Suspension Equilibration Incubation
Regeneration Eluates Wash
Desalting Absorbance
SDS and
Zymography

16. Results

17. Zymography - Enzyme Activity
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
The only eluate that shows activity is
Eluate 1

18. Zymography Gel

19. SDS PAGE – Protein estimation
 SDS PAGE: Protein
estimation
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

20. SDS PAGE
Only the marker and the
HeLa Load can be
appreciated in the gel
image. This might be
result of low
concentration of the rest
of the samples loaded
on to the gel.

21. SDS - PAGE
The second
SDS PAGE
was stained
using silver
stain. This time
elution 1 can
be easily
distinguished.

22. 0
0.5
1
1.5
2
2.5
1.47 1.71 13 16.2 18
Absorbance(562nm)
Sample Volume of each sample (µl)

23. Conclusions
 Plasminogen activator can be effectively
separated from mammalian cell culture.
 The use of magnetic nanoparticles is an
efficient method for protein separation.
 Have an efficient reusability.

24. Acknowledgements
 Dr Vibha Bansal
 RISE Program
 Alexandra Rosado
 Osvaldo Vega
 Natalia Espada
 José J. Rosado
 Mariana León

25. PROTEIN SEPARATION
USING MAGNETIC
NANOPARTICLES
Anthony Maldonado Castro
Félix Vallés
Dr. Vibha Bansal
RISE Program