Optimizing factors for the efficient expression and purification of spao and lamb from Salmonella typhi
Autor(a) principal: | |
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Data de Publicação: | 2022 |
Outros Autores: | , , , , , |
Tipo de documento: | Artigo |
Idioma: | eng |
Título da fonte: | Bioscience journal (Online) |
Texto Completo: | https://seer.ufu.br/index.php/biosciencejournal/article/view/61149 |
Resumo: | We optimized the expression and purification of outer membrane proteins SpaO and LamB from Salmonella typhi. We investigated various factors in the expression and purification processes, including the use of isopropyl β-d-1 thiogalactopyranoside (IPTG), imidazole, and urea. First, PCR amplification was carried out on SpaO and LamB genes. The genes were then cloned in pTZ57R/T, and then expressed in pET28a vector and transformed into Escherichia coli BL21 (DE3). Gene insertion was confirmed by enzymatic digestion with NdeI and XhoI. Inclusion bodies expressing recombinant SpaO and LamB were induced with 200 and 400 µL 0.5 mM IPTG, respectively. The formed protein inclusion bodies were then isolated from the pellet and solubilized in IB buffer containing 8 M urea for SpaO and 6 M urea for LamB. Proteins were refolded by dialysis in 3M urea. Purified proteins with nickel-nitrilotriacetic acid affinity chromatography and eluted with buffer containing 250 mM imidazole for SpaO and 150 mM imidazole for LamB. The protein expression profiles were analyzed by SDS-PAGE, which identified the 33 and 49 kDa bands corresponding to rSpaO and rLamB. Western blotting Purification was carried out by nickel affinity resin with 250 mM and 150 mM imidazole for rSpaO and rLamB and refolded through stepwise dialysis with anti-His tag antibodies confirmed their expression. These optimized methods can be used to generate recombinant proteins for the development of future vaccines. |
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Optimizing factors for the efficient expression and purification of spao and lamb from Salmonella typhi ExpressionOuter Membrane ProteinPurificationSalmonella typhiTyphoid. Biological SciencesWe optimized the expression and purification of outer membrane proteins SpaO and LamB from Salmonella typhi. We investigated various factors in the expression and purification processes, including the use of isopropyl β-d-1 thiogalactopyranoside (IPTG), imidazole, and urea. First, PCR amplification was carried out on SpaO and LamB genes. The genes were then cloned in pTZ57R/T, and then expressed in pET28a vector and transformed into Escherichia coli BL21 (DE3). Gene insertion was confirmed by enzymatic digestion with NdeI and XhoI. Inclusion bodies expressing recombinant SpaO and LamB were induced with 200 and 400 µL 0.5 mM IPTG, respectively. The formed protein inclusion bodies were then isolated from the pellet and solubilized in IB buffer containing 8 M urea for SpaO and 6 M urea for LamB. Proteins were refolded by dialysis in 3M urea. Purified proteins with nickel-nitrilotriacetic acid affinity chromatography and eluted with buffer containing 250 mM imidazole for SpaO and 150 mM imidazole for LamB. The protein expression profiles were analyzed by SDS-PAGE, which identified the 33 and 49 kDa bands corresponding to rSpaO and rLamB. Western blotting Purification was carried out by nickel affinity resin with 250 mM and 150 mM imidazole for rSpaO and rLamB and refolded through stepwise dialysis with anti-His tag antibodies confirmed their expression. These optimized methods can be used to generate recombinant proteins for the development of future vaccines. EDUFU2022-09-23info:eu-repo/semantics/articleinfo:eu-repo/semantics/publishedVersionapplication/pdfhttps://seer.ufu.br/index.php/biosciencejournal/article/view/6114910.14393/BJ-v38n0a2022-61149Bioscience Journal ; Vol. 38 (2022): Continuous Publication; e38084Bioscience Journal ; v. 38 (2022): Continuous Publication; e380841981-3163reponame:Bioscience journal (Online)instname:Universidade Federal de Uberlândia (UFU)instacron:UFUenghttps://seer.ufu.br/index.php/biosciencejournal/article/view/61149/34651Pakistan; Contemporary Copyright (c) 2022 Kunza Latif , Shagufta Naz, Imran Altaf , Jian dong Huang, Rasheeda Bashir, Farheen Aslam , Shaozhen Xinghttps://creativecommons.org/licenses/by/4.0info:eu-repo/semantics/openAccessLatif , KunzaNaz, ShaguftaAltaf , ImranHuang, Jian dongBashir, RasheedaAslam , FarheenShaozhen Xing2022-09-23T15:50:57Zoai:ojs.www.seer.ufu.br:article/61149Revistahttps://seer.ufu.br/index.php/biosciencejournalPUBhttps://seer.ufu.br/index.php/biosciencejournal/oaibiosciencej@ufu.br||1981-31631516-3725opendoar:2022-09-23T15:50:57Bioscience journal (Online) - Universidade Federal de Uberlândia (UFU)false |
dc.title.none.fl_str_mv |
Optimizing factors for the efficient expression and purification of spao and lamb from Salmonella typhi |
title |
Optimizing factors for the efficient expression and purification of spao and lamb from Salmonella typhi |
spellingShingle |
Optimizing factors for the efficient expression and purification of spao and lamb from Salmonella typhi Latif , Kunza Expression Outer Membrane Protein Purification Salmonella typhi Typhoid. Biological Sciences |
title_short |
Optimizing factors for the efficient expression and purification of spao and lamb from Salmonella typhi |
title_full |
Optimizing factors for the efficient expression and purification of spao and lamb from Salmonella typhi |
title_fullStr |
Optimizing factors for the efficient expression and purification of spao and lamb from Salmonella typhi |
title_full_unstemmed |
Optimizing factors for the efficient expression and purification of spao and lamb from Salmonella typhi |
title_sort |
Optimizing factors for the efficient expression and purification of spao and lamb from Salmonella typhi |
author |
Latif , Kunza |
author_facet |
Latif , Kunza Naz, Shagufta Altaf , Imran Huang, Jian dong Bashir, Rasheeda Aslam , Farheen Shaozhen Xing |
author_role |
author |
author2 |
Naz, Shagufta Altaf , Imran Huang, Jian dong Bashir, Rasheeda Aslam , Farheen Shaozhen Xing |
author2_role |
author author author author author author |
dc.contributor.author.fl_str_mv |
Latif , Kunza Naz, Shagufta Altaf , Imran Huang, Jian dong Bashir, Rasheeda Aslam , Farheen Shaozhen Xing |
dc.subject.por.fl_str_mv |
Expression Outer Membrane Protein Purification Salmonella typhi Typhoid. Biological Sciences |
topic |
Expression Outer Membrane Protein Purification Salmonella typhi Typhoid. Biological Sciences |
description |
We optimized the expression and purification of outer membrane proteins SpaO and LamB from Salmonella typhi. We investigated various factors in the expression and purification processes, including the use of isopropyl β-d-1 thiogalactopyranoside (IPTG), imidazole, and urea. First, PCR amplification was carried out on SpaO and LamB genes. The genes were then cloned in pTZ57R/T, and then expressed in pET28a vector and transformed into Escherichia coli BL21 (DE3). Gene insertion was confirmed by enzymatic digestion with NdeI and XhoI. Inclusion bodies expressing recombinant SpaO and LamB were induced with 200 and 400 µL 0.5 mM IPTG, respectively. The formed protein inclusion bodies were then isolated from the pellet and solubilized in IB buffer containing 8 M urea for SpaO and 6 M urea for LamB. Proteins were refolded by dialysis in 3M urea. Purified proteins with nickel-nitrilotriacetic acid affinity chromatography and eluted with buffer containing 250 mM imidazole for SpaO and 150 mM imidazole for LamB. The protein expression profiles were analyzed by SDS-PAGE, which identified the 33 and 49 kDa bands corresponding to rSpaO and rLamB. Western blotting Purification was carried out by nickel affinity resin with 250 mM and 150 mM imidazole for rSpaO and rLamB and refolded through stepwise dialysis with anti-His tag antibodies confirmed their expression. These optimized methods can be used to generate recombinant proteins for the development of future vaccines. |
publishDate |
2022 |
dc.date.none.fl_str_mv |
2022-09-23 |
dc.type.driver.fl_str_mv |
info:eu-repo/semantics/article info:eu-repo/semantics/publishedVersion |
format |
article |
status_str |
publishedVersion |
dc.identifier.uri.fl_str_mv |
https://seer.ufu.br/index.php/biosciencejournal/article/view/61149 10.14393/BJ-v38n0a2022-61149 |
url |
https://seer.ufu.br/index.php/biosciencejournal/article/view/61149 |
identifier_str_mv |
10.14393/BJ-v38n0a2022-61149 |
dc.language.iso.fl_str_mv |
eng |
language |
eng |
dc.relation.none.fl_str_mv |
https://seer.ufu.br/index.php/biosciencejournal/article/view/61149/34651 |
dc.rights.driver.fl_str_mv |
https://creativecommons.org/licenses/by/4.0 info:eu-repo/semantics/openAccess |
rights_invalid_str_mv |
https://creativecommons.org/licenses/by/4.0 |
eu_rights_str_mv |
openAccess |
dc.format.none.fl_str_mv |
application/pdf |
dc.coverage.none.fl_str_mv |
Pakistan; Contemporary |
dc.publisher.none.fl_str_mv |
EDUFU |
publisher.none.fl_str_mv |
EDUFU |
dc.source.none.fl_str_mv |
Bioscience Journal ; Vol. 38 (2022): Continuous Publication; e38084 Bioscience Journal ; v. 38 (2022): Continuous Publication; e38084 1981-3163 reponame:Bioscience journal (Online) instname:Universidade Federal de Uberlândia (UFU) instacron:UFU |
instname_str |
Universidade Federal de Uberlândia (UFU) |
instacron_str |
UFU |
institution |
UFU |
reponame_str |
Bioscience journal (Online) |
collection |
Bioscience journal (Online) |
repository.name.fl_str_mv |
Bioscience journal (Online) - Universidade Federal de Uberlândia (UFU) |
repository.mail.fl_str_mv |
biosciencej@ufu.br|| |
_version_ |
1797069066385489920 |