A set of dual promoter vectors for high throughput cloning, screening, and protein expression in eukaryotic and prokaryotic systems from a single plasmid
<p>Abstract</p> <p>Background</p> <p>The ability to produce the same recombinant protein in both prokaryotic and eukaryotic cells offers many experimental opportunities. However, the cloning of the same gene into multiple plasmids is required, which is time consuming, l...
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| Format: | Article |
| Language: | English |
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BMC
2012-08-01
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| Series: | BMC Biotechnology |
| Online Access: | http://www.biomedcentral.com/1472-6750/12/54 |
| _version_ | 1811314153000796160 |
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| author | Sinah Namita Williams Charlotte A Piper Robert C Shields S |
| author_facet | Sinah Namita Williams Charlotte A Piper Robert C Shields S |
| author_sort | Sinah Namita |
| collection | DOAJ |
| description | <p>Abstract</p> <p>Background</p> <p>The ability to produce the same recombinant protein in both prokaryotic and eukaryotic cells offers many experimental opportunities. However, the cloning of the same gene into multiple plasmids is required, which is time consuming, laborious and still may not produce soluble, stable protein in sufficient quantities. We have developed a set of expression vectors that allows for ligation-independent cloning and rapid functional screening for protein expression in both <it>E. coli</it> and <it>S. cerevisiae.</it></p> <p>Results</p> <p>A set of expression vectors was made that can express the same open reading frame in <it>E. coli</it> (via the T7 phage promoter) and in <it>S. cerevisiae</it> (via the <it>CUP1</it> or <it>MET25</it> promoter). These plasmids also contain the essential elements for replication and selection in both cell types and have several advantages: they allow for cloning of genes by homologous recombination in yeast, protein expression can be determined before plasmid isolation and sequencing, and a GST-fusion tag is added to aid in soluble expression and purification. We have also included a TEV recognition site that allows for the specific cleavage of the fusion proteins to yield native proteins.</p> <p>Conclusions</p> <p>The dual promoter vectors can be used for rapid cloning, expression, and purification of target proteins from both prokaryotic and eukaryotic systems with the ability to study post-translation modifications.</p> |
| first_indexed | 2024-04-13T11:06:48Z |
| format | Article |
| id | doaj.art-a570868f4c74400db8e9467f5eb5c634 |
| institution | Directory Open Access Journal |
| issn | 1472-6750 |
| language | English |
| last_indexed | 2024-04-13T11:06:48Z |
| publishDate | 2012-08-01 |
| publisher | BMC |
| record_format | Article |
| series | BMC Biotechnology |
| spelling | doaj.art-a570868f4c74400db8e9467f5eb5c6342022-12-22T02:49:14ZengBMCBMC Biotechnology1472-67502012-08-011215410.1186/1472-6750-12-54A set of dual promoter vectors for high throughput cloning, screening, and protein expression in eukaryotic and prokaryotic systems from a single plasmidSinah NamitaWilliams Charlotte APiper Robert CShields S<p>Abstract</p> <p>Background</p> <p>The ability to produce the same recombinant protein in both prokaryotic and eukaryotic cells offers many experimental opportunities. However, the cloning of the same gene into multiple plasmids is required, which is time consuming, laborious and still may not produce soluble, stable protein in sufficient quantities. We have developed a set of expression vectors that allows for ligation-independent cloning and rapid functional screening for protein expression in both <it>E. coli</it> and <it>S. cerevisiae.</it></p> <p>Results</p> <p>A set of expression vectors was made that can express the same open reading frame in <it>E. coli</it> (via the T7 phage promoter) and in <it>S. cerevisiae</it> (via the <it>CUP1</it> or <it>MET25</it> promoter). These plasmids also contain the essential elements for replication and selection in both cell types and have several advantages: they allow for cloning of genes by homologous recombination in yeast, protein expression can be determined before plasmid isolation and sequencing, and a GST-fusion tag is added to aid in soluble expression and purification. We have also included a TEV recognition site that allows for the specific cleavage of the fusion proteins to yield native proteins.</p> <p>Conclusions</p> <p>The dual promoter vectors can be used for rapid cloning, expression, and purification of target proteins from both prokaryotic and eukaryotic systems with the ability to study post-translation modifications.</p>http://www.biomedcentral.com/1472-6750/12/54 |
| spellingShingle | Sinah Namita Williams Charlotte A Piper Robert C Shields S A set of dual promoter vectors for high throughput cloning, screening, and protein expression in eukaryotic and prokaryotic systems from a single plasmid BMC Biotechnology |
| title | A set of dual promoter vectors for high throughput cloning, screening, and protein expression in eukaryotic and prokaryotic systems from a single plasmid |
| title_full | A set of dual promoter vectors for high throughput cloning, screening, and protein expression in eukaryotic and prokaryotic systems from a single plasmid |
| title_fullStr | A set of dual promoter vectors for high throughput cloning, screening, and protein expression in eukaryotic and prokaryotic systems from a single plasmid |
| title_full_unstemmed | A set of dual promoter vectors for high throughput cloning, screening, and protein expression in eukaryotic and prokaryotic systems from a single plasmid |
| title_short | A set of dual promoter vectors for high throughput cloning, screening, and protein expression in eukaryotic and prokaryotic systems from a single plasmid |
| title_sort | set of dual promoter vectors for high throughput cloning screening and protein expression in eukaryotic and prokaryotic systems from a single plasmid |
| url | http://www.biomedcentral.com/1472-6750/12/54 |
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