Cloning, Expression and Purification of Subunit H of Vacuolar H+-ATPase from Mythimna separata Walker (Lepidoptera: Noctuidae)
The vacuolar (H+)-ATPase (V-ATPase) of insect, which is composed of membrane-bound V0 complex and peripheral V1 complex, participates in lots of important physiological process. Subunit H, as a subunit of V1 complex, plays a vital role in bridging the communication between V1 and V0 complexes and in...
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MDPI AG
2014-09-01
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| Series: | International Journal of Molecular Sciences |
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| Online Access: | http://www.mdpi.com/1422-0067/15/9/15443 |
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| author | Lina Lu Zhijun Qi Wenjun Wu |
| author_facet | Lina Lu Zhijun Qi Wenjun Wu |
| author_sort | Lina Lu |
| collection | DOAJ |
| description | The vacuolar (H+)-ATPase (V-ATPase) of insect, which is composed of membrane-bound V0 complex and peripheral V1 complex, participates in lots of important physiological process. Subunit H, as a subunit of V1 complex, plays a vital role in bridging the communication between V1 and V0 complexes and interaction with other proteins. Yeast subunit H has been successfully crystallized through expression in E. coli, but little is known about the structure of insect subunit H. In this study, we cloned, expressed and purified the subunit H from midgut of Mythimna separata Walker. Through RACE (rapidly amplification of cDNA ends) technique, we got 1807 bp full length of subunit H, and to keep the nature structure of subunit H, we constructed Baculovirus expression vector with His-tag in the C-terminal and expressed the recombinant protein in insect sf9 cells, thereafter, purified the recombinant protein by Ni-NTA columns. Results of SDS-PAGE, western blotting and mass spectrometry showed that the recombinant protein was successfully expressed. The method of expressing and purifying M. separata subunit H will provide a foundation for obtaining the crystal of subunit H and further study of the design of novel insecticides based on its structure and function. |
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| institution | Directory Open Access Journal |
| issn | 1422-0067 |
| language | English |
| last_indexed | 2024-04-12T03:06:10Z |
| publishDate | 2014-09-01 |
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| spelling | doaj.art-4d663d662b314892bfd0e72fa26cf4e52022-12-22T03:50:30ZengMDPI AGInternational Journal of Molecular Sciences1422-00672014-09-01159154431545510.3390/ijms150915443ijms150915443Cloning, Expression and Purification of Subunit H of Vacuolar H+-ATPase from Mythimna separata Walker (Lepidoptera: Noctuidae)Lina Lu0Zhijun Qi1Wenjun Wu2Institute of Pesticide Science, Northwest Agriculture and Forestry University, Yangling 712100, Shaanxi, ChinaInstitute of Pesticide Science, Northwest Agriculture and Forestry University, Yangling 712100, Shaanxi, ChinaInstitute of Pesticide Science, Northwest Agriculture and Forestry University, Yangling 712100, Shaanxi, ChinaThe vacuolar (H+)-ATPase (V-ATPase) of insect, which is composed of membrane-bound V0 complex and peripheral V1 complex, participates in lots of important physiological process. Subunit H, as a subunit of V1 complex, plays a vital role in bridging the communication between V1 and V0 complexes and interaction with other proteins. Yeast subunit H has been successfully crystallized through expression in E. coli, but little is known about the structure of insect subunit H. In this study, we cloned, expressed and purified the subunit H from midgut of Mythimna separata Walker. Through RACE (rapidly amplification of cDNA ends) technique, we got 1807 bp full length of subunit H, and to keep the nature structure of subunit H, we constructed Baculovirus expression vector with His-tag in the C-terminal and expressed the recombinant protein in insect sf9 cells, thereafter, purified the recombinant protein by Ni-NTA columns. Results of SDS-PAGE, western blotting and mass spectrometry showed that the recombinant protein was successfully expressed. The method of expressing and purifying M. separata subunit H will provide a foundation for obtaining the crystal of subunit H and further study of the design of novel insecticides based on its structure and function.http://www.mdpi.com/1422-0067/15/9/15443vacuolar (H+)-ATPasesMythimna separatasubunit Hinsect expression system |
| spellingShingle | Lina Lu Zhijun Qi Wenjun Wu Cloning, Expression and Purification of Subunit H of Vacuolar H+-ATPase from Mythimna separata Walker (Lepidoptera: Noctuidae) International Journal of Molecular Sciences vacuolar (H+)-ATPases Mythimna separata subunit H insect expression system |
| title | Cloning, Expression and Purification of Subunit H of Vacuolar H+-ATPase from Mythimna separata Walker (Lepidoptera: Noctuidae) |
| title_full | Cloning, Expression and Purification of Subunit H of Vacuolar H+-ATPase from Mythimna separata Walker (Lepidoptera: Noctuidae) |
| title_fullStr | Cloning, Expression and Purification of Subunit H of Vacuolar H+-ATPase from Mythimna separata Walker (Lepidoptera: Noctuidae) |
| title_full_unstemmed | Cloning, Expression and Purification of Subunit H of Vacuolar H+-ATPase from Mythimna separata Walker (Lepidoptera: Noctuidae) |
| title_short | Cloning, Expression and Purification of Subunit H of Vacuolar H+-ATPase from Mythimna separata Walker (Lepidoptera: Noctuidae) |
| title_sort | cloning expression and purification of subunit h of vacuolar h atpase from mythimna separata walker lepidoptera noctuidae |
| topic | vacuolar (H+)-ATPases Mythimna separata subunit H insect expression system |
| url | http://www.mdpi.com/1422-0067/15/9/15443 |
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