Cloning, Expression, and in vitro Functional Activity Assay of phiC31 Integrase cDNA in Escherichia coli

OBJECTIVE: The aim of present study was cloning and expression of phiC31 integrase cDNA in a bacterial expression vector. Thus, an intra molecular assay vector was applied to show in vitro activity of recombinant protein. MATERIALS AND METHODS: In this experimental study, phiC31 cDNA was subcloned i...

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Autores principales: Sekhavati, Mohammad Hadi, Tahmoorespur, Mojtaba, Ghaedi, Kamran, Dormiani, Kianoush, Nassiri, Mohammad Reza, Khazaie, Yahya, Foruzanfar, Mahboubeh, Hosseini, Morteza, Nasr Esfahani, Mohammad Hossein
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Royan Institute 2013
Materias:
Research Article
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3593930/
https://www.ncbi.nlm.nih.gov/pubmed/23577305
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author Sekhavati, Mohammad Hadi
Tahmoorespur, Mojtaba
Ghaedi, Kamran
Dormiani, Kianoush
Nassiri, Mohammad Reza
Khazaie, Yahya
Foruzanfar, Mahboubeh
Hosseini, Morteza
Nasr Esfahani, Mohammad Hossein
author_facet Sekhavati, Mohammad Hadi
Tahmoorespur, Mojtaba
Ghaedi, Kamran
Dormiani, Kianoush
Nassiri, Mohammad Reza
Khazaie, Yahya
Foruzanfar, Mahboubeh
Hosseini, Morteza
Nasr Esfahani, Mohammad Hossein
author_sort Sekhavati, Mohammad Hadi
collection PubMed
description OBJECTIVE: The aim of present study was cloning and expression of phiC31 integrase cDNA in a bacterial expression vector. Thus, an intra molecular assay vector was applied to show in vitro activity of recombinant protein. MATERIALS AND METHODS: In this experimental study, phiC31 cDNA was subcloned into a prokaryotic expression vector and transformed into E.coli Bl21 (DE3). Recombinant phiC31 integrase was purified form the bacterial cell lysates and its activity was verified by an in vitro functional assessment. RESULTS: Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of the purified phiC31 integrase confirmed the size of protein (70 kDa). Finally, the functionality of purified phiC31 integrase was verified. CONCLUSION: The results of this study indicated that the purified integrase has a great potential application for in vitro site-specific integration.
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spelling pubmed-35939302013-04-10 Cloning, Expression, and in vitro Functional Activity Assay of phiC31 Integrase cDNA in Escherichia coli Sekhavati, Mohammad Hadi Tahmoorespur, Mojtaba Ghaedi, Kamran Dormiani, Kianoush Nassiri, Mohammad Reza Khazaie, Yahya Foruzanfar, Mahboubeh Hosseini, Morteza Nasr Esfahani, Mohammad Hossein Cell J Research Article OBJECTIVE: The aim of present study was cloning and expression of phiC31 integrase cDNA in a bacterial expression vector. Thus, an intra molecular assay vector was applied to show in vitro activity of recombinant protein. MATERIALS AND METHODS: In this experimental study, phiC31 cDNA was subcloned into a prokaryotic expression vector and transformed into E.coli Bl21 (DE3). Recombinant phiC31 integrase was purified form the bacterial cell lysates and its activity was verified by an in vitro functional assessment. RESULTS: Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of the purified phiC31 integrase confirmed the size of protein (70 kDa). Finally, the functionality of purified phiC31 integrase was verified. CONCLUSION: The results of this study indicated that the purified integrase has a great potential application for in vitro site-specific integration. Royan Institute 2013 2013-02-20 /pmc/articles/PMC3593930/ /pubmed/23577305 Text en Any use, distribution, reproduction or abstract of this publication in any medium, with the exception of commercial purposes, is permitted provided the original work is properly cited http://creativecommons.org/licenses/by/2.5/ This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
spellingShingle Research Article
Sekhavati, Mohammad Hadi
Tahmoorespur, Mojtaba
Ghaedi, Kamran
Dormiani, Kianoush
Nassiri, Mohammad Reza
Khazaie, Yahya
Foruzanfar, Mahboubeh
Hosseini, Morteza
Nasr Esfahani, Mohammad Hossein
Cloning, Expression, and in vitro Functional Activity Assay of phiC31 Integrase cDNA in Escherichia coli
title Cloning, Expression, and in vitro Functional Activity Assay of phiC31 Integrase cDNA in Escherichia coli
title_full Cloning, Expression, and in vitro Functional Activity Assay of phiC31 Integrase cDNA in Escherichia coli
title_fullStr Cloning, Expression, and in vitro Functional Activity Assay of phiC31 Integrase cDNA in Escherichia coli
title_full_unstemmed Cloning, Expression, and in vitro Functional Activity Assay of phiC31 Integrase cDNA in Escherichia coli
title_short Cloning, Expression, and in vitro Functional Activity Assay of phiC31 Integrase cDNA in Escherichia coli
title_sort cloning, expression, and in vitro functional activity assay of phic31 integrase cdna in escherichia coli
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3593930/
https://www.ncbi.nlm.nih.gov/pubmed/23577305
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