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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 V(0) complex and peripheral V(1) complex, participates in lots of important physiological process. Subunit H, as a subunit of V(1) complex, plays a vital role in bridging the communication between V(1) and V(0) comp...

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Autores principales: Lu, Lina, Qi, Zhijun, Wu, Wenjun
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2014
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4200773/
https://www.ncbi.nlm.nih.gov/pubmed/25257524
http://dx.doi.org/10.3390/ijms150915443
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author Lu, Lina
Qi, Zhijun
Wu, Wenjun
author_facet Lu, Lina
Qi, Zhijun
Wu, Wenjun
author_sort Lu, Lina
collection PubMed
description The vacuolar (H(+))-ATPase (V-ATPase) of insect, which is composed of membrane-bound V(0) complex and peripheral V(1) complex, participates in lots of important physiological process. Subunit H, as a subunit of V(1) complex, plays a vital role in bridging the communication between V(1) and V(0) 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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spelling pubmed-42007732014-10-17 Cloning, Expression and Purification of Subunit H of Vacuolar H(+)-ATPase from Mythimna separata Walker (Lepidoptera: Noctuidae) Lu, Lina Qi, Zhijun Wu, Wenjun Int J Mol Sci Article The vacuolar (H(+))-ATPase (V-ATPase) of insect, which is composed of membrane-bound V(0) complex and peripheral V(1) complex, participates in lots of important physiological process. Subunit H, as a subunit of V(1) complex, plays a vital role in bridging the communication between V(1) and V(0) 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. MDPI 2014-09-01 /pmc/articles/PMC4200773/ /pubmed/25257524 http://dx.doi.org/10.3390/ijms150915443 Text en © 2014 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution license (http://creativecommons.org/licenses/by/3.0/).
spellingShingle Article
Lu, Lina
Qi, Zhijun
Wu, Wenjun
Cloning, Expression and Purification of Subunit H of Vacuolar H(+)-ATPase from Mythimna separata Walker (Lepidoptera: Noctuidae)
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 Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4200773/
https://www.ncbi.nlm.nih.gov/pubmed/25257524
http://dx.doi.org/10.3390/ijms150915443
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