This study characterized the recombinant DNA polymerase beta (polβ) enzyme from Trypanosoma cruzi. Researchers expressed and purified the polβ gene from the T. cruzi Miranda clone. Assays showed the purified recombinant enzyme had DNA polymerase activity. Western blots confirmed the protein was polβ using generated antibodies. Analysis also found the native form of T. cruzi polβ is phosphorylated. DNA repair assays demonstrated the recombinant enzyme can repair damaged DNA. This work helps understand the role of polβ in T. cruzi, which could aid in developing new drug targets for Chagas disease.

1. Expression, purification, and biochemical characterization
of recombinant DNA polymerase beta of the Trypanosoma cruzi
TcI lineage: requirement of additional factors and detection
of phosphorylation of the native form
Edio Maldonado, Diego A. Rojas, Sandra Moreira-Ramos, Fabiola
Urbina , Vicente J. Miralles, Aldo Solari & Juan Venegas
Michelle Larios Gómez
Maria Alejandra Galeano
Teacher: Lina Maria Martinez
III Semester
Medicine Faculty

2. INTRODUCTION
Chagas disease is a debilitating condition cause by infection of
the protozoan parasite trypanosoma cruzi. Aproximately 20
million people in Latin America are infected with the parasite and
50,000 deaths anually are asociated with the infection.
 Genus Trypanosoma
 Species Cruzi

3. TRYPANOSOMA
 Trypanosoma is a genus of kinetoplastids, a
monophyletic group of unicellular parasitic flagellate
protozoa.
 All trypanosomes are heteroxenous and most are
transmitted via a vector.

4. TRYPANOSOMA CRUZI
Transmission  Hematophagous insect vector.
 Congenital infection
 Ingestion of food contaminated
with infective forms of the parasite

5. DIAGNOSIS
 CONVENTIONAL
1. Detection of trypanosomes in fresh It is the most simple
and consists of
examining a drop of
blood to the
microscope
2. Strout's concentration method It consists in the
observation of parasites in
the sediment of the blood
serum after centrifugation

6. DIAGNOSIS
3. Peripheral blood smear
Thin layer of blood smeared on a
microscope slide and then stained to
allow the various blood cells to be
examined microscopically.

7. DIAGNOSIS
 MOLECULAR
1. Enzyme-linked immunosorbent assay (ELISA):
Laboratory technique that allows to detect antigens
To be able to identify the antigens are used molecules with
two connected components: an antibody and an enzyme.
The enzyme is activated and
indicates the union to the antigen
by a change of color

8. DIAGNOSIS
Technique of immunolabeling that
makes use of antibodies
chemically united to a fluorescent
substance to demonstrate the
presence of a specific molecule
2. Indirect Immunofluorescence
Is based on the property that the
antibodies have to produce specific
agglutination in the presence of red blood
cells sensitized with the corresponding
antigens.
3. Indirect hemoagglutination

9. TRYPANOSOMA CRUZI
KINETOPLAST
Network of circular DNA inside a
large mitochondrion that contains
many copies of the mitochondrial
genome
In kinetoplast replication,
the DNA polymerase beta
(polß) has a key function in
DNA repair

10. OBJECTIVE
Show the expression of the polß gene in a highly efficient
bacterial expression vector, purification of the enzyme, and a
study of the biochemical characterization of the recombinant
DNA polymerase ß encoded by the T. cruzi Miranda clone
(Miranda Tcpolß), which belongs to the TcI lineage.

11. MATERIALES Y METODOS
Plásmido
puc-72
Plantilla que
contiene
tcpolβ
Amplificado con
PCR:
1) 5 min a 94º
2) 55º
3) 1 min a 72º
4) 10 min a 72º
Vector fácil:
pGEM-T
Vector de
expresión:
pET15b
Bacterias
DH5α
PCR

12. EXPRESION Y PURIFICACION DE LA
TCPOLβ RECOMBINANTE
1. Medio de cultivo: platos de agar de luria bertali
2. Se las incubo con terrific broth
3. Se indujeron usando IPTG
4. Se centrifugan y se suspenden en un buffer
5. Se separan proteínas de cuerpos insolubles
6. Las proteínas se diluyen se dializan y se purifican con MI-NTA
7. Purificación final de la polimerasa recombinante por
intercambio iónico

13. El rendimiento de la purificación de la
polimerasa fue de 45 a 50 mg,
obtenida a partir de 500 ml
SDS- PAGE Y WESTERN
BLOT
EXPRESION Y PURIFICACION DE LA
TCPOLβ RECOMBINANTE
Figura 1a

14. ENSAYO ENZIMATICO
Se llevo a cabo con 5 microlitros de la fracción de hidroxiapatita
en un buffer reactivo obteniendo una actividad especifica de
438,24 cmp/pmol en un vol total de ensayo de 50 microlitros
Se incubo  papel de DE81  lavaron
CONTADOR DE CENTELLEO
Actividad: nucleótidos/mg de proteína

15. ESPECTOMETRIA DE MASAS
1. Se escindió la banda de la proteína del SDS-PAGE
2. Se ioniza y se hace impacto electrónico
3. Colector/analizador  masa/ carga
4. Se analizan con cromatografía liquida
= CORRESPONDEN A TCPOLβ

16. GENERACION DE
ANTICUERPOS
Se inmunizo por varios días con
la proteína recombinante de
diferentes formas unos conejos
para después mediante la
obtención de muestras de sangre
se extraerán y purificaran los
anticuerpos

17. GENERACION DE
ANTICUERPOS
Figura 1b

18. PREPARACION DE LOS
EXTRACTOS DE PROTEINA DE
T.CRUZI
• Las células se suspendieron en un buffer con el fin
de lisarlas
• Se incubaron para posteriormente centrifugarlas
= por medio de diálisis se extrajeron las
proteínas

19. PURIFICACIÓN DE LA TCPOLβ
ASOCIADA A PROTEÍNAS
Luego de la extracción de las proteínas se procede
a purificar la enzima separándola de las proteínas
Para esto se utilizaron anticuerpos buffers y una
resina que permite la separación

20. ANÁLISIS DE
FOSFOPROTEÍNAS
Este procedimiento realizo con el fin de comprobar la
existencia de fosfatos en la forma nativa de la enzima
SDS-PAGE

21. ENSAYO DE REPARACIÓN
DE DNA
A partir de un kit de ensayo de polimerasa ya
fabricada (short gap filling kit) se realizo la prueba de
reparación de DNA con y sin la adición de tcpolβ.

22. RESULTADOS
Figura 1

23. Figura 5
RESULTADOS

24. RESULTADOS
Figura 6

25. Figura 7
RESULTADOS

26. DISCUSSION
AUTHOR PHRASE YES NO
Venegas et al. “Despite the similarity of the TcI polß
Miranda clone to the CL Brener clone,
they are not identical”
Almeida and Sobol “At least 16 proteins have been described
that form a complex with polß; among
them it is important to mention XRCC1
and PARP1, which interact directly with
polß and appear to be responsible for
forming a kind of molecular skeleton for
complex stability”

27. DISCUSSION
AUTHOR PHRASE YES NO
(Lopes et al;
Schamber-Reis et
al.)
“Although there are other recombinant
Tcpolß described in the literature; they
are from the CL Brener clone of the TcVI
lineage which were expressed as fusion
peptides bound to maltose-binding
protein”
Stalker et al. “In some cases, the mammalian polßs
are highly sensitive to NEM, such as the
polß from the Novikoff hepatoma”

28. CONCLUSIONS
 This study helped us to understand more the role
and biological characteristics of the polymerase beta
in the parasite and this would be a great advance in
the development of treatments for Chagas disease
 There are no effective drugs for the chronic phase of
the Chagas disease so understanding the similarities
and differences with the mammal polymerase could
help us to identify more easily chemo-therapeutic
targets

29. CONCLUSIONS
 Based on this study now we know the
characteristics of the polymerase enzyme that is
also found in mammals and which has the ability
to repair damaged DNA, providing survival
advantages to trypanosoma cruzi
 With the help of these molecular methods the
different genetic components involved in the
operation and development of parasites that affect
the community can be characterized

30. CONCLUSIONES

31. CONCLUSIONES

32. BIBLIOGRAPHY
 Molecular mechanism of Chagas disease: Lymphocyte activation by
the trypanosoma cruzi, Wenda Gao
 http://www.minsalud.gov.co/Documentos%20y%20Publicaciones/Gu
ia%20de%20atencion%20clinica%20de%20chagas%202010.pdf
 Botero, David; Restrepo, Marcos. Parasitosis humanas. 5. ed.
Medellín: Corporación para Investigaciones Biológicas. 2012.