Expression of a Haemonchus contortus cysteine protease in the baculovirus system
Electron. j. biotechnol
;
11(2): 49-55, Apr. 2008. ilus, graf, tab
Artigo
em Inglês
| LILACS
| ID: lil-522205
ABSTRACT
A Haemonchus contortus recombinant Cysteine Protease (CP) was expressed in the baculovirus system. The CP gene was isolated by PCR from H. contortus cDNA, the PCR amplicon was cloned downstream to the polihedrin promoter within a bacterial expression vector, Sf9 insect cells were used for simultaneous co-transfection with the CP-vector and baculovirus naked DNA, which originated recombinant viruses by homologous recombination capable to express recombinant CP in an insect cell culture. A recombinant protease was identified as a fusion protein with a Ni lithium affinity 6XHis group. Recombinant CP was purified by affinity chromatography to obtain active recombinant protease identified by H. contortus experimentally infested ovine sera on a western blot as a 37 kDa protein, as well as by enzyme activity on PAGE-gelatin. Cysteine protease activity was assayed against synthetic substrates including the dipeptides Phe-Arg, cathepsin B substrate Arg-Arg, the caspase tetrapeptide substrate Tyr-Val-Ala-Asp. Maximum CP activity was detected at pH 6.0 for all synthetic substrates and total inhibition was achieved by E-64 but not by EDTA, pepstatin or PMSF. Recombinant H. contortus CP can be obtained in large amounts from transfected insect cell culture and may be applied to control experiments of ruminant Haemonchosis.
Texto completo:
DisponíveL
Índice:
LILACS (Américas)
Assunto principal:
Cisteína Endopeptidases
/
Baculoviridae
/
Haemonchus
Idioma:
Inglês
Revista:
Electron. j. biotechnol
Assunto da revista:
Biotecnologia
Ano de publicação:
2008
Tipo de documento:
Artigo
/
Documento de projeto
País de afiliação:
México
Instituição/País de afiliação:
Centro Nacional de Investigación Disciplinaria en Parasitología Veterinaria/MX
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