Expression of a Haemonchus contortus cysteine protease in the baculovirus system

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 simultaneo...

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Autores principales:	Miranda-Miranda,Estefan, Murillo-Sánchez,María Hortensia, Cossío-Bayúgar,Raquel
Lenguaje:	English
Publicado:	Pontificia Universidad Católica de Valparaíso 2008
Materias:	haemonchosis molecular cloning recombinant protease synthetic substrates western blot
Acceso en línea:	http://www.scielo.cl/scielo.php?script=sci_arttext&pid=S0717-34582008000200007
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spelling	oai:scielo:S0717-345820080002000072009-01-21Expression of a Haemonchus contortus cysteine protease in the baculovirus systemMiranda-Miranda,EstefanMurillo-Sánchez,María HortensiaCossío-Bayúgar,Raquel haemonchosis molecular cloning recombinant protease synthetic substrates western blot 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.info:eu-repo/semantics/openAccessPontificia Universidad Católica de ValparaísoElectronic Journal of Biotechnology v.11 n.2 20082008-04-01text/htmlhttp://www.scielo.cl/scielo.php?script=sci_arttext&pid=S0717-34582008000200007en10.4067/S0717-34582008000200007
institution	Scielo Chile
collection	Scielo Chile
language	English
topic	haemonchosis molecular cloning recombinant protease synthetic substrates western blot
spellingShingle	haemonchosis molecular cloning recombinant protease synthetic substrates western blot Miranda-Miranda,Estefan Murillo-Sánchez,María Hortensia Cossío-Bayúgar,Raquel Expression of a Haemonchus contortus cysteine protease in the baculovirus system
description	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.
author	Miranda-Miranda,Estefan Murillo-Sánchez,María Hortensia Cossío-Bayúgar,Raquel
author_facet	Miranda-Miranda,Estefan Murillo-Sánchez,María Hortensia Cossío-Bayúgar,Raquel
author_sort	Miranda-Miranda,Estefan
title	Expression of a Haemonchus contortus cysteine protease in the baculovirus system
title_short	Expression of a Haemonchus contortus cysteine protease in the baculovirus system
title_full	Expression of a Haemonchus contortus cysteine protease in the baculovirus system
title_fullStr	Expression of a Haemonchus contortus cysteine protease in the baculovirus system
title_full_unstemmed	Expression of a Haemonchus contortus cysteine protease in the baculovirus system
title_sort	expression of a haemonchus contortus cysteine protease in the baculovirus system
publisher	Pontificia Universidad Católica de Valparaíso
publishDate	2008
url	http://www.scielo.cl/scielo.php?script=sci_arttext&pid=S0717-34582008000200007
work_keys_str_mv	AT mirandamirandaestefan expressionofahaemonchuscontortuscysteineproteaseinthebaculovirussystem AT murillosanchezmariahortensia expressionofahaemonchuscontortuscysteineproteaseinthebaculovirussystem AT cossiobayugarraquel expressionofahaemonchuscontortuscysteineproteaseinthebaculovirussystem
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Expression of a Haemonchus contortus cysteine protease in the baculovirus system

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