Efficient Expression and Purification of Recombinant Human Enteropeptidase Light Chain in Esherichia coli
Autor(a) principal: | |
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Data de Publicação: | 2015 |
Outros Autores: | , , |
Tipo de documento: | Artigo |
Idioma: | eng |
Título da fonte: | Brazilian Archives of Biology and Technology |
Texto Completo: | http://old.scielo.br/scielo.php?script=sci_arttext&pid=S1516-89132015000200154 |
Resumo: | Human enterokinase (synonym: enteropeptidase, EC 3.4.21.9) light chain (hEKL) gene was designed and artificially synthesized with built-in codon blas towards Escherichia colicodon preference. The synthetic hEKL gene was cloned into prokaryotic expression vector pMAL-s and transferred into the expression strain E. coli BL21 (DE3). Recombinant hEKL protein with a maltose binding protein (MBP) tag was expressed at high levels in soluble form, which yielded about 42% of the total cellular protein. The target protein was then purified to the homogeneity (> 95%) by affinity chromatography. The peptide substrate GST-Melittin with enterokinase recognition site was completely cleaved by the purified MBP-hEKL at the molar ratio of 1:5000 (enzyme:substrate). Tricine SDS-PAGE analysis showed that the activity of MBP-hEKL was approximately seven times that of bovine enterokinase catalytic subunit (EKMaxTM, Invitrogen). From 1 L flask culture, 206 mg pure active MBP-hEKL was with specific activity of 1.4×104U/mg. |
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Efficient Expression and Purification of Recombinant Human Enteropeptidase Light Chain in Esherichia coliHuman enterokinase light chain (hEKL)Fusion expressionAffinity purificationActivity analysisEnzymatic cleavageHuman enterokinase (synonym: enteropeptidase, EC 3.4.21.9) light chain (hEKL) gene was designed and artificially synthesized with built-in codon blas towards Escherichia colicodon preference. The synthetic hEKL gene was cloned into prokaryotic expression vector pMAL-s and transferred into the expression strain E. coli BL21 (DE3). Recombinant hEKL protein with a maltose binding protein (MBP) tag was expressed at high levels in soluble form, which yielded about 42% of the total cellular protein. The target protein was then purified to the homogeneity (> 95%) by affinity chromatography. The peptide substrate GST-Melittin with enterokinase recognition site was completely cleaved by the purified MBP-hEKL at the molar ratio of 1:5000 (enzyme:substrate). Tricine SDS-PAGE analysis showed that the activity of MBP-hEKL was approximately seven times that of bovine enterokinase catalytic subunit (EKMaxTM, Invitrogen). From 1 L flask culture, 206 mg pure active MBP-hEKL was with specific activity of 1.4×104U/mg.Instituto de Tecnologia do Paraná - Tecpar2015-04-01info:eu-repo/semantics/articleinfo:eu-repo/semantics/publishedVersiontext/htmlhttp://old.scielo.br/scielo.php?script=sci_arttext&pid=S1516-89132015000200154Brazilian Archives of Biology and Technology v.58 n.2 2015reponame:Brazilian Archives of Biology and Technologyinstname:Instituto de Tecnologia do Paraná (Tecpar)instacron:TECPAR10.1590/S1516-8913201400094info:eu-repo/semantics/openAccessNiu,Li-XiLi,Jia-YueJi,Xue-XueYang,Bin-Shengeng2015-10-08T00:00:00Zoai:scielo:S1516-89132015000200154Revistahttps://www.scielo.br/j/babt/https://old.scielo.br/oai/scielo-oai.phpbabt@tecpar.br||babt@tecpar.br1678-43241516-8913opendoar:2015-10-08T00:00Brazilian Archives of Biology and Technology - Instituto de Tecnologia do Paraná (Tecpar)false |
dc.title.none.fl_str_mv |
Efficient Expression and Purification of Recombinant Human Enteropeptidase Light Chain in Esherichia coli |
title |
Efficient Expression and Purification of Recombinant Human Enteropeptidase Light Chain in Esherichia coli |
spellingShingle |
Efficient Expression and Purification of Recombinant Human Enteropeptidase Light Chain in Esherichia coli Niu,Li-Xi Human enterokinase light chain (hEKL) Fusion expression Affinity purification Activity analysis Enzymatic cleavage |
title_short |
Efficient Expression and Purification of Recombinant Human Enteropeptidase Light Chain in Esherichia coli |
title_full |
Efficient Expression and Purification of Recombinant Human Enteropeptidase Light Chain in Esherichia coli |
title_fullStr |
Efficient Expression and Purification of Recombinant Human Enteropeptidase Light Chain in Esherichia coli |
title_full_unstemmed |
Efficient Expression and Purification of Recombinant Human Enteropeptidase Light Chain in Esherichia coli |
title_sort |
Efficient Expression and Purification of Recombinant Human Enteropeptidase Light Chain in Esherichia coli |
author |
Niu,Li-Xi |
author_facet |
Niu,Li-Xi Li,Jia-Yue Ji,Xue-Xue Yang,Bin-Sheng |
author_role |
author |
author2 |
Li,Jia-Yue Ji,Xue-Xue Yang,Bin-Sheng |
author2_role |
author author author |
dc.contributor.author.fl_str_mv |
Niu,Li-Xi Li,Jia-Yue Ji,Xue-Xue Yang,Bin-Sheng |
dc.subject.por.fl_str_mv |
Human enterokinase light chain (hEKL) Fusion expression Affinity purification Activity analysis Enzymatic cleavage |
topic |
Human enterokinase light chain (hEKL) Fusion expression Affinity purification Activity analysis Enzymatic cleavage |
description |
Human enterokinase (synonym: enteropeptidase, EC 3.4.21.9) light chain (hEKL) gene was designed and artificially synthesized with built-in codon blas towards Escherichia colicodon preference. The synthetic hEKL gene was cloned into prokaryotic expression vector pMAL-s and transferred into the expression strain E. coli BL21 (DE3). Recombinant hEKL protein with a maltose binding protein (MBP) tag was expressed at high levels in soluble form, which yielded about 42% of the total cellular protein. The target protein was then purified to the homogeneity (> 95%) by affinity chromatography. The peptide substrate GST-Melittin with enterokinase recognition site was completely cleaved by the purified MBP-hEKL at the molar ratio of 1:5000 (enzyme:substrate). Tricine SDS-PAGE analysis showed that the activity of MBP-hEKL was approximately seven times that of bovine enterokinase catalytic subunit (EKMaxTM, Invitrogen). From 1 L flask culture, 206 mg pure active MBP-hEKL was with specific activity of 1.4×104U/mg. |
publishDate |
2015 |
dc.date.none.fl_str_mv |
2015-04-01 |
dc.type.driver.fl_str_mv |
info:eu-repo/semantics/article |
dc.type.status.fl_str_mv |
info:eu-repo/semantics/publishedVersion |
format |
article |
status_str |
publishedVersion |
dc.identifier.uri.fl_str_mv |
http://old.scielo.br/scielo.php?script=sci_arttext&pid=S1516-89132015000200154 |
url |
http://old.scielo.br/scielo.php?script=sci_arttext&pid=S1516-89132015000200154 |
dc.language.iso.fl_str_mv |
eng |
language |
eng |
dc.relation.none.fl_str_mv |
10.1590/S1516-8913201400094 |
dc.rights.driver.fl_str_mv |
info:eu-repo/semantics/openAccess |
eu_rights_str_mv |
openAccess |
dc.format.none.fl_str_mv |
text/html |
dc.publisher.none.fl_str_mv |
Instituto de Tecnologia do Paraná - Tecpar |
publisher.none.fl_str_mv |
Instituto de Tecnologia do Paraná - Tecpar |
dc.source.none.fl_str_mv |
Brazilian Archives of Biology and Technology v.58 n.2 2015 reponame:Brazilian Archives of Biology and Technology instname:Instituto de Tecnologia do Paraná (Tecpar) instacron:TECPAR |
instname_str |
Instituto de Tecnologia do Paraná (Tecpar) |
instacron_str |
TECPAR |
institution |
TECPAR |
reponame_str |
Brazilian Archives of Biology and Technology |
collection |
Brazilian Archives of Biology and Technology |
repository.name.fl_str_mv |
Brazilian Archives of Biology and Technology - Instituto de Tecnologia do Paraná (Tecpar) |
repository.mail.fl_str_mv |
babt@tecpar.br||babt@tecpar.br |
_version_ |
1750318276851793920 |