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23 Congress of the International Union for Biochemistry and Molecular Biology
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44 Annual Meeting of the Brazilian Society for Biochemistry and Molecular Biology
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Foz do Iguaçu, PR, Brazil, August 24 to 28 , 2015
WHOLE GENOME SHOTGUN PHAGE-DISPLAY FOR THE DE NOVO
DISCOVERY OF TRYPANOSOMA CRUZI AND HOST CELL INTERACTIONS
André A. R. Teixeira1, Fabiana L. Ferreira2, Paulo Lee Ho2, Walter Colli1, Maria Júlia M.
Alves1, Ricardo J. Giordano1
1
Departamento de Bioquímica, Instituto de Química, Universidade de São Paulo, São Paulo, SP, Brazil;
2
Centro de Biotecnologia, Instituto Butantan, São Paulo, SP, Brazil
Introduction and Objectives: Trypanosoma cruzi is a protozoan parasite causative of
Chagas disease. During its life cycle, the parasite in its non-replicative trypomastigote
form has to recognize, attach and invade cells of the mammalian host in order to
escape the immune system and to replicate. The invasion is a complex biochemical
process where many adhesion and signaling molecules are involved. Our goal is to
discover new T.cruzi proteins that are important for host cell recognition and invasion.
To achieve our aim, we are using the phage display technology. This technique enables
the display of proteins fused to the viral capsid and the selection of high affinity ligands
to specific targets in a process called biopanning. We have taken advantage of the fact
that T.cruzi genes do not contain introns to build a shotgun library using the parasite
genomic DNA. Thus, the resulting phage particles express T.cruzi proteins on its
surface. This library may now be used to select parasite proteins with high affinity to
host molecules and help improve our understanding of parasite-host interactions.
Material and methods: The phage display library was built using the phagemid vector
pG8SAET. The genomic DNA of the Sylvio X10 strain was sheared using Covaris S2 to
produce fragments of 100-500 bp, which were blunt ligated to the linearized vector.
Results: We have successfully built a library containing 4.4x108 phage clones with an
average insert size of 254 bp (about 85 amino acids). The total library size (254 bp x
4.4x108 = 1.1x1011 bp) covers the haploid genome (4.4x107 bp) more than 2,500 times.
Conclusions. We have built a phage display library using the genomic DNA of the
parasite T.cruzi. Given the high coverage we obtained, the library should probably
include all proteins encoded in the parasite genome and be suitable for the discovery of
new proteins that participate in host cell interaction.
Key Words. Trypanosoma cruzi; Phage Display, Protein-protein interactions.
Brazilian Society for Biochemistry and
Molecular Biology (SBBq)