Expression, purification and characterization of refolded rBm-33 (pepsin inhibitor homolog) from Brugia malayi: a human Lymphatic Filarial parasite.
Identifieur interne : 004516 ( Ncbi/Checkpoint ); précédent : 004515; suivant : 004517Expression, purification and characterization of refolded rBm-33 (pepsin inhibitor homolog) from Brugia malayi: a human Lymphatic Filarial parasite.
Auteurs : Nagampalli Raghavendra Sashi Krishna [Inde] ; N S A. Krushna ; R B Narayanan ; S S Rajan ; K. GunasekaranSource :
- Protein expression and purification [ 1096-0279 ] ; 2011.
Descripteurs français
- KwdFr :
- Animaux, Brugia malayi (enzymologie), Brugia malayi (génétique), Caséines (métabolisme), Chromatographie sur gel, Cinétique, Clonage moléculaire (), Corps d'inclusion (), Corps d'inclusion (métabolisme), Dichroïsme circulaire, Escherichia coli, Filariose lymphatique (métabolisme), Filariose lymphatique (parasitologie), Glutaraldéhyde (), Humains, Inhibiteurs de protéases (isolement et purification), Inhibiteurs de protéases (métabolisme), Inhibiteurs de protéases (pharmacologie), Pepsine A (antagonistes et inhibiteurs), Pepsine A (métabolisme), Plasmides, Protéines recombinantes (isolement et purification), Protéines recombinantes (métabolisme), Protéines recombinantes (pharmacologie), Repliement des protéines, Structure secondaire des protéines, Technique de Western, Transformation bactérienne.
- MESH :
- antagonistes et inhibiteurs : Pepsine A.
- enzymologie : Brugia malayi.
- génétique : Brugia malayi.
- isolement et purification : Inhibiteurs de protéases, Protéines recombinantes.
- métabolisme : Caséines, Corps d'inclusion, Filariose lymphatique, Inhibiteurs de protéases, Pepsine A, Protéines recombinantes.
- parasitologie : Filariose lymphatique.
- pharmacologie : Inhibiteurs de protéases, Protéines recombinantes.
- Animaux, Chromatographie sur gel, Cinétique, Clonage moléculaire, Corps d'inclusion, Dichroïsme circulaire, Escherichia coli, Glutaraldéhyde, Humains, Plasmides, Repliement des protéines, Structure secondaire des protéines, Technique de Western, Transformation bactérienne.
English descriptors
- KwdEn :
- Animals, Blotting, Western, Brugia malayi (enzymology), Brugia malayi (genetics), Caseins (metabolism), Chromatography, Gel, Circular Dichroism, Cloning, Molecular (methods), Elephantiasis, Filarial (metabolism), Elephantiasis, Filarial (parasitology), Escherichia coli, Glutaral (chemistry), Humans, Inclusion Bodies (chemistry), Inclusion Bodies (metabolism), Kinetics, Pepsin A (antagonists & inhibitors), Pepsin A (metabolism), Plasmids, Protease Inhibitors (isolation & purification), Protease Inhibitors (metabolism), Protease Inhibitors (pharmacology), Protein Refolding, Protein Structure, Secondary, Recombinant Proteins (isolation & purification), Recombinant Proteins (metabolism), Recombinant Proteins (pharmacology), Transformation, Bacterial.
- MESH :
- chemical , antagonists & inhibitors : Pepsin A.
- chemical , chemistry : Glutaral.
- chemical , isolation & purification : Protease Inhibitors, Recombinant Proteins.
- chemical , metabolism : Caseins, Pepsin A, Protease Inhibitors, Recombinant Proteins.
- chemistry : Inclusion Bodies.
- enzymology : Brugia malayi.
- genetics : Brugia malayi.
- metabolism : Elephantiasis, Filarial, Inclusion Bodies.
- methods : Cloning, Molecular.
- parasitology : Elephantiasis, Filarial.
- chemical , pharmacology : Protease Inhibitors, Recombinant Proteins.
- Animals, Blotting, Western, Chromatography, Gel, Circular Dichroism, Escherichia coli, Humans, Kinetics, Plasmids, Protein Refolding, Protein Structure, Secondary, Transformation, Bacterial.
Abstract
Bm-33 (pepsin inhibitor homolog) produced by the human filarial parasite Brugia malayi, was expressed in Escherichia coli. Expression of rBm33 in BL21 (DE3), Rosetta-2 gami (DE3) pLysS and GJ1158 bacterial strains, results in the accumulation of a 33 kDa protein in inclusion bodies. Inactive rBm-33 was purified under the denaturing conditions and refolded by step wise dialysis using buffers of pH ranging from 11 to 7. Size exclusion chromatography of rBm-33 (refolded) reveals that nearly 83% of the recombinant protein exhibits pepsin inhibition activity. Circular dichroism studies indicate that the protein is predominantly composed of 85% α-helix. rBm-33 (refolded) was assessed for its pepsin inhibition activity using casein agar plate method, UV-spectroscopy and zymogram analysis. These findings suggest that rBm-33 (refolded) has affinity for human pepsin and completely inhibits the proteolytic activity with the gradual increase in rBm-33 (refolded) concentration. Size exclusion chromatography reveals the formation of rBm-33-pepsin complex and was cross checked using immunoblot with glutaraldehyde cross linking. These findings reveal that rBm-33 (refolded) is in native fold to exhibit pepsin inhibition.
DOI: 10.1016/j.pep.2011.06.014
PubMed: 21745575
Affiliations:
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Krushna</name></author><author><name sortKey="Narayanan, R B" sort="Narayanan, R B" uniqKey="Narayanan R" first="R B" last="Narayanan">R B Narayanan</name></author><author><name sortKey="Rajan, S S" sort="Rajan, S S" uniqKey="Rajan S" first="S S" last="Rajan">S S Rajan</name></author><author><name sortKey="Gunasekaran, K" sort="Gunasekaran, K" uniqKey="Gunasekaran K" first="K" last="Gunasekaran">K. Gunasekaran</name></author></analytic><series><title level="j">Protein expression and purification</title><idno type="eISSN">1096-0279</idno><imprint><date when="2011" type="published">2011</date></imprint></series></biblStruct></sourceDesc></fileDesc><profileDesc><textClass><keywords scheme="KwdEn" xml:lang="en"><term>Animals</term><term>Blotting, Western</term><term>Brugia malayi (enzymology)</term><term>Brugia malayi (genetics)</term><term>Caseins (metabolism)</term><term>Chromatography, Gel</term><term>Circular Dichroism</term><term>Cloning, Molecular (methods)</term><term>Elephantiasis, Filarial (metabolism)</term><term>Elephantiasis, Filarial (parasitology)</term><term>Escherichia coli</term><term>Glutaral (chemistry)</term><term>Humans</term><term>Inclusion Bodies (chemistry)</term><term>Inclusion Bodies (metabolism)</term><term>Kinetics</term><term>Pepsin A (antagonists & inhibitors)</term><term>Pepsin A (metabolism)</term><term>Plasmids</term><term>Protease Inhibitors (isolation & purification)</term><term>Protease Inhibitors (metabolism)</term><term>Protease Inhibitors (pharmacology)</term><term>Protein Refolding</term><term>Protein Structure, Secondary</term><term>Recombinant Proteins (isolation & purification)</term><term>Recombinant Proteins (metabolism)</term><term>Recombinant Proteins (pharmacology)</term><term>Transformation, Bacterial</term></keywords><keywords scheme="KwdFr" xml:lang="fr"><term>Animaux</term><term>Brugia malayi (enzymologie)</term><term>Brugia malayi (génétique)</term><term>Caséines (métabolisme)</term><term>Chromatographie sur gel</term><term>Cinétique</term><term>Clonage moléculaire ()</term><term>Corps d'inclusion ()</term><term>Corps d'inclusion (métabolisme)</term><term>Dichroïsme circulaire</term><term>Escherichia coli</term><term>Filariose lymphatique (métabolisme)</term><term>Filariose lymphatique (parasitologie)</term><term>Glutaraldéhyde ()</term><term>Humains</term><term>Inhibiteurs de protéases (isolement et purification)</term><term>Inhibiteurs de protéases (métabolisme)</term><term>Inhibiteurs de protéases (pharmacologie)</term><term>Pepsine A (antagonistes et inhibiteurs)</term><term>Pepsine A (métabolisme)</term><term>Plasmides</term><term>Protéines recombinantes (isolement et purification)</term><term>Protéines recombinantes (métabolisme)</term><term>Protéines recombinantes (pharmacologie)</term><term>Repliement des protéines</term><term>Structure secondaire des protéines</term><term>Technique de Western</term><term>Transformation bactérienne</term></keywords><keywords scheme="MESH" type="chemical" qualifier="antagonists & inhibitors" xml:lang="en"><term>Pepsin A</term></keywords><keywords scheme="MESH" type="chemical" qualifier="chemistry" xml:lang="en"><term>Glutaral</term></keywords><keywords scheme="MESH" type="chemical" qualifier="isolation & purification" xml:lang="en"><term>Protease Inhibitors</term><term>Recombinant Proteins</term></keywords><keywords scheme="MESH" type="chemical" qualifier="metabolism" xml:lang="en"><term>Caseins</term><term>Pepsin A</term><term>Protease Inhibitors</term><term>Recombinant Proteins</term></keywords><keywords scheme="MESH" qualifier="antagonistes et inhibiteurs" xml:lang="fr"><term>Pepsine A</term></keywords><keywords scheme="MESH" qualifier="chemistry" xml:lang="en"><term>Inclusion Bodies</term></keywords><keywords scheme="MESH" qualifier="enzymologie" xml:lang="fr"><term>Brugia malayi</term></keywords><keywords scheme="MESH" qualifier="enzymology" xml:lang="en"><term>Brugia malayi</term></keywords><keywords scheme="MESH" qualifier="genetics" xml:lang="en"><term>Brugia malayi</term></keywords><keywords scheme="MESH" qualifier="génétique" xml:lang="fr"><term>Brugia malayi</term></keywords><keywords scheme="MESH" qualifier="isolement et purification" xml:lang="fr"><term>Inhibiteurs de protéases</term><term>Protéines recombinantes</term></keywords><keywords scheme="MESH" qualifier="metabolism" xml:lang="en"><term>Elephantiasis, Filarial</term><term>Inclusion Bodies</term></keywords><keywords scheme="MESH" qualifier="methods" xml:lang="en"><term>Cloning, Molecular</term></keywords><keywords scheme="MESH" qualifier="métabolisme" xml:lang="fr"><term>Caséines</term><term>Corps d'inclusion</term><term>Filariose lymphatique</term><term>Inhibiteurs de protéases</term><term>Pepsine A</term><term>Protéines recombinantes</term></keywords><keywords scheme="MESH" qualifier="parasitologie" xml:lang="fr"><term>Filariose lymphatique</term></keywords><keywords scheme="MESH" qualifier="parasitology" xml:lang="en"><term>Elephantiasis, Filarial</term></keywords><keywords scheme="MESH" qualifier="pharmacologie" xml:lang="fr"><term>Inhibiteurs de protéases</term><term>Protéines recombinantes</term></keywords><keywords scheme="MESH" type="chemical" qualifier="pharmacology" xml:lang="en"><term>Protease Inhibitors</term><term>Recombinant Proteins</term></keywords><keywords scheme="MESH" xml:lang="en"><term>Animals</term><term>Blotting, Western</term><term>Chromatography, Gel</term><term>Circular Dichroism</term><term>Escherichia coli</term><term>Humans</term><term>Kinetics</term><term>Plasmids</term><term>Protein Refolding</term><term>Protein Structure, Secondary</term><term>Transformation, Bacterial</term></keywords><keywords scheme="MESH" xml:lang="fr"><term>Animaux</term><term>Chromatographie sur gel</term><term>Cinétique</term><term>Clonage moléculaire</term><term>Corps d'inclusion</term><term>Dichroïsme circulaire</term><term>Escherichia coli</term><term>Glutaraldéhyde</term><term>Humains</term><term>Plasmides</term><term>Repliement des protéines</term><term>Structure secondaire des protéines</term><term>Technique de Western</term><term>Transformation bactérienne</term></keywords></textClass></profileDesc></teiHeader><front><div type="abstract" xml:lang="en">Bm-33 (pepsin inhibitor homolog) produced by the human filarial parasite Brugia malayi, was expressed in Escherichia coli. Expression of rBm33 in BL21 (DE3), Rosetta-2 gami (DE3) pLysS and GJ1158 bacterial strains, results in the accumulation of a 33 kDa protein in inclusion bodies. Inactive rBm-33 was purified under the denaturing conditions and refolded by step wise dialysis using buffers of pH ranging from 11 to 7. Size exclusion chromatography of rBm-33 (refolded) reveals that nearly 83% of the recombinant protein exhibits pepsin inhibition activity. Circular dichroism studies indicate that the protein is predominantly composed of 85% α-helix. rBm-33 (refolded) was assessed for its pepsin inhibition activity using casein agar plate method, UV-spectroscopy and zymogram analysis. These findings suggest that rBm-33 (refolded) has affinity for human pepsin and completely inhibits the proteolytic activity with the gradual increase in rBm-33 (refolded) concentration. Size exclusion chromatography reveals the formation of rBm-33-pepsin complex and was cross checked using immunoblot with glutaraldehyde cross linking. These findings reveal that rBm-33 (refolded) is in native fold to exhibit pepsin inhibition.</div></front></TEI><affiliations><list><country><li>Inde</li></country></list><tree><noCountry><name sortKey="Gunasekaran, K" sort="Gunasekaran, K" uniqKey="Gunasekaran K" first="K" last="Gunasekaran">K. Gunasekaran</name><name sortKey="Krushna, N S A" sort="Krushna, N S A" uniqKey="Krushna N" first="N S A" last="Krushna">N S A. Krushna</name><name sortKey="Narayanan, R B" sort="Narayanan, R B" uniqKey="Narayanan R" first="R B" last="Narayanan">R B Narayanan</name><name sortKey="Rajan, S S" sort="Rajan, S S" uniqKey="Rajan S" first="S S" last="Rajan">S S Rajan</name></noCountry><country name="Inde"><noRegion><name sortKey="Krishna, Nagampalli Raghavendra Sashi" sort="Krishna, Nagampalli Raghavendra Sashi" uniqKey="Krishna N" first="Nagampalli Raghavendra Sashi" last="Krishna">Nagampalli Raghavendra Sashi Krishna</name></noRegion></country></tree></affiliations></record>Pour manipuler ce document sous Unix (Dilib)
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