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Int. J. Mol. Sci. 2014, 15(9), 15443-15455; doi:10.3390/ijms150915443

Cloning, Expression and Purification of Subunit H of Vacuolar H+-ATPase from Mythimna separata Walker (Lepidoptera: Noctuidae)

Institute of Pesticide Science, Northwest Agriculture and Forestry University, Yangling 712100, Shaanxi, China
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Received: 11 June 2014 / Revised: 17 August 2014 / Accepted: 26 August 2014 / Published: 1 September 2014
(This article belongs to the Section Biochemistry, Molecular and Cellular Biology)
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Abstract

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. View Full-Text
Keywords: vacuolar (H+)-ATPases; Mythimna separata; subunit H; insect expression system vacuolar (H+)-ATPases; Mythimna separata; subunit H; insect expression system
This is an open access article distributed under the Creative Commons Attribution License (CC BY 3.0).

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MDPI and ACS Style

Lu, L.; Qi, Z.; Wu, W. Cloning, Expression and Purification of Subunit H of Vacuolar H+-ATPase from Mythimna separata Walker (Lepidoptera: Noctuidae). Int. J. Mol. Sci. 2014, 15, 15443-15455.

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