Tagging the expressed protein with 6 histidines: rapid cloning of an amplicon with three options.
We report the designing of three expression vectors that can be used for rapid cloning of any blunt-end DNA segment. Only a single set of oligonucleotides are required to perform the amplification of the target DNA and its cloning in all three vectors simultaneously. The DNA thus cloned can express...
| Main Authors: | , |
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| Format: | Article |
| Language: | English |
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Public Library of Science (PLoS)
2013-01-01
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| Series: | PLoS ONE |
| Online Access: | http://europepmc.org/articles/PMC3655076?pdf=render |
| _version_ | 1818574408285945856 |
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| author | Manika Indrajit Singh Vikas Jain |
| author_facet | Manika Indrajit Singh Vikas Jain |
| author_sort | Manika Indrajit Singh |
| collection | DOAJ |
| description | We report the designing of three expression vectors that can be used for rapid cloning of any blunt-end DNA segment. Only a single set of oligonucleotides are required to perform the amplification of the target DNA and its cloning in all three vectors simultaneously. The DNA thus cloned can express a protein either with or without a hexa-histidine tag depending upon the vector used. The expression occurs from T7 promoter when transformed into E. coli BL21(DE3). Two of the three plasmids have been designed to provide the expressed protein with either N- or C-terminus 6 histidine amino acids in tandem. The third plasmid, however, does not add any tag to the expressed protein. The cloning is achieved quickly with the requirement of phosphorylation of PCR product without any restriction digestion. Additionally, the generated clones can be confirmed with a single step PCR reaction carried out from bacterial colonies (generally termed as "colony PCR"). We show the cloning, expression and purification of Green Fluorescent Protein (GFP) as proof-of-concept. Additionally, we also show the cloning and expression of four sigma factors from Mycobacterium tuberculosis further demonstrating the utility of the designed plasmids. We strongly believe that the vectors and the strategy that we have developed will facilitate the rapid cloning and expression of any gene in E. coli BL21(DE3) with or without a hexa-histidine tag. |
| first_indexed | 2024-12-15T00:26:15Z |
| format | Article |
| id | doaj.art-e5c418be84f54a89819e3fc70d3144f0 |
| institution | Directory Open Access Journal |
| issn | 1932-6203 |
| language | English |
| last_indexed | 2024-12-15T00:26:15Z |
| publishDate | 2013-01-01 |
| publisher | Public Library of Science (PLoS) |
| record_format | Article |
| series | PLoS ONE |
| spelling | doaj.art-e5c418be84f54a89819e3fc70d3144f02022-12-21T22:42:09ZengPublic Library of Science (PLoS)PLoS ONE1932-62032013-01-0185e6392210.1371/journal.pone.0063922Tagging the expressed protein with 6 histidines: rapid cloning of an amplicon with three options.Manika Indrajit SinghVikas JainWe report the designing of three expression vectors that can be used for rapid cloning of any blunt-end DNA segment. Only a single set of oligonucleotides are required to perform the amplification of the target DNA and its cloning in all three vectors simultaneously. The DNA thus cloned can express a protein either with or without a hexa-histidine tag depending upon the vector used. The expression occurs from T7 promoter when transformed into E. coli BL21(DE3). Two of the three plasmids have been designed to provide the expressed protein with either N- or C-terminus 6 histidine amino acids in tandem. The third plasmid, however, does not add any tag to the expressed protein. The cloning is achieved quickly with the requirement of phosphorylation of PCR product without any restriction digestion. Additionally, the generated clones can be confirmed with a single step PCR reaction carried out from bacterial colonies (generally termed as "colony PCR"). We show the cloning, expression and purification of Green Fluorescent Protein (GFP) as proof-of-concept. Additionally, we also show the cloning and expression of four sigma factors from Mycobacterium tuberculosis further demonstrating the utility of the designed plasmids. We strongly believe that the vectors and the strategy that we have developed will facilitate the rapid cloning and expression of any gene in E. coli BL21(DE3) with or without a hexa-histidine tag.http://europepmc.org/articles/PMC3655076?pdf=render |
| spellingShingle | Manika Indrajit Singh Vikas Jain Tagging the expressed protein with 6 histidines: rapid cloning of an amplicon with three options. PLoS ONE |
| title | Tagging the expressed protein with 6 histidines: rapid cloning of an amplicon with three options. |
| title_full | Tagging the expressed protein with 6 histidines: rapid cloning of an amplicon with three options. |
| title_fullStr | Tagging the expressed protein with 6 histidines: rapid cloning of an amplicon with three options. |
| title_full_unstemmed | Tagging the expressed protein with 6 histidines: rapid cloning of an amplicon with three options. |
| title_short | Tagging the expressed protein with 6 histidines: rapid cloning of an amplicon with three options. |
| title_sort | tagging the expressed protein with 6 histidines rapid cloning of an amplicon with three options |
| url | http://europepmc.org/articles/PMC3655076?pdf=render |
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