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A modified Gateway cloning strategy for overexpressing tagged proteins in plants
BACKGROUND: Recent developments, including the sequencing of a number of plant genomes, have greatly increased the amount of data available to scientists and has enabled high throughput investigations where many genes are investigated simultaneously. To perform these studies, recombinational cloning...
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Formato: | Texto |
Lenguaje: | English |
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BioMed Central
2008
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Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2267177/ https://www.ncbi.nlm.nih.gov/pubmed/18211686 http://dx.doi.org/10.1186/1746-4811-4-3 |
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author | Dubin, Manu J Bowler, Chris Benvenuto, Giovanna |
author_facet | Dubin, Manu J Bowler, Chris Benvenuto, Giovanna |
author_sort | Dubin, Manu J |
collection | PubMed |
description | BACKGROUND: Recent developments, including the sequencing of a number of plant genomes, have greatly increased the amount of data available to scientists and has enabled high throughput investigations where many genes are investigated simultaneously. To perform these studies, recombinational cloning methods such as the Gateway system have been adapted to plant transformation vectors to facilitate the creation of overexpression, tagging and silencing vectors on a large scale. RESULTS: Here we present a hybrid cloning strategy which combines advantages of both recombinational and traditional cloning and which is particularly amenable to low-to-medium throughput investigations of protein function using techniques of molecular biochemistry and cell biology. The system consists of a series of twelve Gateway Entry cassettes into which a gene of interest can be inserted using traditional cloning methods to generate either N- or C-terminal fusions to epitope tags and fluorescent proteins. The resulting gene-tag fusions can then be recombined into Gateway-based Destination vectors, thus providing a wide choice of resistance marker, promoter and expression system. The advantage of this modified Gateway cloning strategy is that the entire open reading frame encoding the tagged protein of interest is contained within the Entry vectors so that after recombination no additional linker sequences are added between the tag and the protein that could interfere with protein function and expression. We demonstrate the utility of this system for both transient and stable Agrobacterium-mediated plant transformations. CONCLUSION: This modified Gateway cloning strategy is complementary to more conventional Gateway-based systems because it expands the choice of tags and higher orders of combinations, and permits more control over the linker sequence lying between a protein of interest and an epitope tag, which can be particularly important for studies of protein function. |
format | Text |
id | pubmed-2267177 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2008 |
publisher | BioMed Central |
record_format | MEDLINE/PubMed |
spelling | pubmed-22671772008-03-13 A modified Gateway cloning strategy for overexpressing tagged proteins in plants Dubin, Manu J Bowler, Chris Benvenuto, Giovanna Plant Methods Research BACKGROUND: Recent developments, including the sequencing of a number of plant genomes, have greatly increased the amount of data available to scientists and has enabled high throughput investigations where many genes are investigated simultaneously. To perform these studies, recombinational cloning methods such as the Gateway system have been adapted to plant transformation vectors to facilitate the creation of overexpression, tagging and silencing vectors on a large scale. RESULTS: Here we present a hybrid cloning strategy which combines advantages of both recombinational and traditional cloning and which is particularly amenable to low-to-medium throughput investigations of protein function using techniques of molecular biochemistry and cell biology. The system consists of a series of twelve Gateway Entry cassettes into which a gene of interest can be inserted using traditional cloning methods to generate either N- or C-terminal fusions to epitope tags and fluorescent proteins. The resulting gene-tag fusions can then be recombined into Gateway-based Destination vectors, thus providing a wide choice of resistance marker, promoter and expression system. The advantage of this modified Gateway cloning strategy is that the entire open reading frame encoding the tagged protein of interest is contained within the Entry vectors so that after recombination no additional linker sequences are added between the tag and the protein that could interfere with protein function and expression. We demonstrate the utility of this system for both transient and stable Agrobacterium-mediated plant transformations. CONCLUSION: This modified Gateway cloning strategy is complementary to more conventional Gateway-based systems because it expands the choice of tags and higher orders of combinations, and permits more control over the linker sequence lying between a protein of interest and an epitope tag, which can be particularly important for studies of protein function. BioMed Central 2008-01-22 /pmc/articles/PMC2267177/ /pubmed/18211686 http://dx.doi.org/10.1186/1746-4811-4-3 Text en Copyright © 2008 Dubin et al; licensee BioMed Central Ltd. http://creativecommons.org/licenses/by/2.0 This is an Open Access article distributed under the terms of the Creative Commons Attribution License ( (http://creativecommons.org/licenses/by/2.0) ), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. |
spellingShingle | Research Dubin, Manu J Bowler, Chris Benvenuto, Giovanna A modified Gateway cloning strategy for overexpressing tagged proteins in plants |
title | A modified Gateway cloning strategy for overexpressing tagged proteins in plants |
title_full | A modified Gateway cloning strategy for overexpressing tagged proteins in plants |
title_fullStr | A modified Gateway cloning strategy for overexpressing tagged proteins in plants |
title_full_unstemmed | A modified Gateway cloning strategy for overexpressing tagged proteins in plants |
title_short | A modified Gateway cloning strategy for overexpressing tagged proteins in plants |
title_sort | modified gateway cloning strategy for overexpressing tagged proteins in plants |
topic | Research |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2267177/ https://www.ncbi.nlm.nih.gov/pubmed/18211686 http://dx.doi.org/10.1186/1746-4811-4-3 |
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