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

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

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.

TRYPANOSOMA CRUZI

Transmission Hematophagous insect vector.

Congenital infection

Ingestion of food contaminated

with infective forms of the parasite

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

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.

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

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

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

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.

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

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

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

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

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

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

GENERACION DE

ANTICUERPOS

Figura 1b

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

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

ANÁLISIS DE

FOSFOPROTEÍNAS

Este procedimiento realizo con el fin de comprobar la

existencia de fosfatos en la forma nativa de la enzima

SDS-PAGE

ENSAYO DE REPARACIÓN

DE DNA

A partir de un kit de ensayo de polimerasa ya

fabricada (short gap filling kit) se realizo la prueba dereparación de DNA con y sin la adición de tcpolβ.

RESULTADOS

Figura 1

Figura 5

RESULTADOS

RESULTADOS

Figura 6

Figura 7

RESULTADOS

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”

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”

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

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

CONCLUSIONES

CONCLUSIONES

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.