Enzymatic characterization of a recombinant aspartic proteinase from Boophilus microplus
Pohl, P.C.1; Leal, A.T.1; Sorgine, M.4; Medrano, F.J.5; Vaz, I.1, 2; Masuda, A.1, 3
1 Centro de Biotecnologia-UFRGS; 2 Fac. Veterinaria-UFRGS; 3 Depto Biologia Molecular e Biotecnologia-UFRGS; 4 Depto Bioquímica. Médica-UFRJ; 5 Laboratório Nacional de Luz Síncrontron
The tick Boophilus microplus
is a bovine hematophagous ectoparasite, present in tropical and
subtropical areas in the world. The actual method for the control of
cattle tick is the use of chemical pesticides, which causes damage to
the environment and contaminates the meat and milk. An alternative and
promising method for control is the use of vaccines. This work is aimed
towards the identification of new proteins that have immunoprotective
potential. THAP (tick heme-binding aspartic proteinase) is an aspartic
proteinase present in eggs of B. microplus involved in
the embryogenesis, by degrading vitellin according with the necessity
of embryonic development. We have cloned the full protein sequence
after restoring of the four initial amino acids of the THAP. It was
necessary since initial nucleotides of the exon were absent in the
original cDNA. Through PCR it was obtained an amplicon with 1065 bp
that was cloned into the pET43a expression vector. The resultant
plasmid (pET43a-THAPr) was transformed by electroporation in eleven Escherichia coli strains
BL21 (DE3). The best conditions established for production of the
recombinant protein (THAPr with fusion protein Nus-Tag) in the soluble
form was the expression in E.coli BL21 (DE3) RIL at 23ºC and 1 mM
of IPTG for 4 hours. Analysis of the expression was performed by
SDS-PAGE and Western-blot using a polyclonal serum anti-THAP. For the
purification of THAPr was used an affinity chromatography column with
sepharose-Ni2+. After purification the fusion protein,
Nus-Tag, was removed. A fraction partially purified was obtained and
its enzymatic activity was assayed with the fluorogenic substrate
Abz-AIAFFSRQ-EDDnp. The enzymatic activity was monitored for 1 hour
with an F-MAX fluorometer and the specific activity obtained was 9.55
RFU/min/mg of protein. Its activity was blocked by 20 µM of pepstatin
A. Further studies on the THAP enzymatic activity and its potential
immunoprotective role are in progress to define the importance of this
protein for tick control.
This work was supported by grants from CNPq- PIBIC, PRONEX- FAPERJ
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