Recombinant expression library of Pyrococcus furiosus constructed by high-throughput cloning: a useful tool for functional and structural genomics
Hyperthermophile Pyrococcus furiosus grows optimally near 100°C and is an important resource of many industrial and molecular biological enzymes. To study the structure and function of Pyrococcus furiosus proteins at whole genome level, we constructed expression plasmids of each Pyrococcus furiosus...
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| Format: | Article |
| Language: | English |
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Frontiers Media S.A.
2015-09-01
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| Series: | Frontiers in Microbiology |
| Subjects: | |
| Online Access: | http://journal.frontiersin.org/Journal/10.3389/fmicb.2015.00943/full |
| _version_ | 1828885057859747840 |
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| author | Hui eYuan Li ePeng Zhong eHan Juan-Juan eXie Xi-Peng eLiu |
| author_facet | Hui eYuan Li ePeng Zhong eHan Juan-Juan eXie Xi-Peng eLiu |
| author_sort | Hui eYuan |
| collection | DOAJ |
| description | Hyperthermophile Pyrococcus furiosus grows optimally near 100°C and is an important resource of many industrial and molecular biological enzymes. To study the structure and function of Pyrococcus furiosus proteins at whole genome level, we constructed expression plasmids of each Pyrococcus furiosus gene using a ligase-independent cloning method, which was based on amplifying target gene and vector by PCR using phosphorothioate-modified primers and digesting PCR products by λ exonuclease. Our cloning method had a positive clone percentage of ≥ 80% in 96-well plate cloning format. Small-scale expression experiment showed that 55 out of 80 genes were efficiently expressed in Escherichia coli Strain Rosetta 2(DE3)pLysS. In summary, this recombinant expression library of Pyrococcus furiosus provides a platform for functional and structural studies, as well as developing novel industrial enzymes. Our cloning scheme is adaptable to constructing recombinant expression library of other sequenced organisms. |
| first_indexed | 2024-12-13T11:18:23Z |
| format | Article |
| id | doaj.art-40df6f400d4746efad87d6fed95ca572 |
| institution | Directory Open Access Journal |
| issn | 1664-302X |
| language | English |
| last_indexed | 2024-12-13T11:18:23Z |
| publishDate | 2015-09-01 |
| publisher | Frontiers Media S.A. |
| record_format | Article |
| series | Frontiers in Microbiology |
| spelling | doaj.art-40df6f400d4746efad87d6fed95ca5722022-12-21T23:48:32ZengFrontiers Media S.A.Frontiers in Microbiology1664-302X2015-09-01610.3389/fmicb.2015.00943157036Recombinant expression library of Pyrococcus furiosus constructed by high-throughput cloning: a useful tool for functional and structural genomicsHui eYuan0Li ePeng1Zhong eHan2Juan-Juan eXie3Xi-Peng eLiu4Shanghai Jiao Tong UniversityShanghai Jiao Tong UniversityShanghai Jiao Tong UniversityShanghai Jiao Tong UniversityShanghai Jiao Tong UniversityHyperthermophile Pyrococcus furiosus grows optimally near 100°C and is an important resource of many industrial and molecular biological enzymes. To study the structure and function of Pyrococcus furiosus proteins at whole genome level, we constructed expression plasmids of each Pyrococcus furiosus gene using a ligase-independent cloning method, which was based on amplifying target gene and vector by PCR using phosphorothioate-modified primers and digesting PCR products by λ exonuclease. Our cloning method had a positive clone percentage of ≥ 80% in 96-well plate cloning format. Small-scale expression experiment showed that 55 out of 80 genes were efficiently expressed in Escherichia coli Strain Rosetta 2(DE3)pLysS. In summary, this recombinant expression library of Pyrococcus furiosus provides a platform for functional and structural studies, as well as developing novel industrial enzymes. Our cloning scheme is adaptable to constructing recombinant expression library of other sequenced organisms.http://journal.frontiersin.org/Journal/10.3389/fmicb.2015.00943/fullPyrococcus furiosusLigation-independent cloningrecombinant expression libraryhigh-throughput cloningphosphorothioate modification. |
| spellingShingle | Hui eYuan Li ePeng Zhong eHan Juan-Juan eXie Xi-Peng eLiu Recombinant expression library of Pyrococcus furiosus constructed by high-throughput cloning: a useful tool for functional and structural genomics Frontiers in Microbiology Pyrococcus furiosus Ligation-independent cloning recombinant expression library high-throughput cloning phosphorothioate modification. |
| title | Recombinant expression library of Pyrococcus furiosus constructed by high-throughput cloning: a useful tool for functional and structural genomics |
| title_full | Recombinant expression library of Pyrococcus furiosus constructed by high-throughput cloning: a useful tool for functional and structural genomics |
| title_fullStr | Recombinant expression library of Pyrococcus furiosus constructed by high-throughput cloning: a useful tool for functional and structural genomics |
| title_full_unstemmed | Recombinant expression library of Pyrococcus furiosus constructed by high-throughput cloning: a useful tool for functional and structural genomics |
| title_short | Recombinant expression library of Pyrococcus furiosus constructed by high-throughput cloning: a useful tool for functional and structural genomics |
| title_sort | recombinant expression library of pyrococcus furiosus constructed by high throughput cloning a useful tool for functional and structural genomics |
| topic | Pyrococcus furiosus Ligation-independent cloning recombinant expression library high-throughput cloning phosphorothioate modification. |
| url | http://journal.frontiersin.org/Journal/10.3389/fmicb.2015.00943/full |
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