title: Expression, purification and characterization of human vacuolar-type H(+)-ATPase subunit d1 and d2 in Escherichia coli.
abstract: Vacuolar-type H(+)-ATPase (V-ATPase) is a multi-subunit proton pump. The proton pump is essential for the regulation of pH in various eukaryotic cellular processes. Among the 14 subunits that constitute V-ATPase, d subunit mediates coupling between cytosolic and membrane domains. Whereas d1 is expressed ubiquitously in various types of cells, its isoform d2 is only expressed in specific cells or tissues. To characterize these isoforms, we expressed and purified the isoforms of human V-ATPase d subunits using Escherichia coli over-expression system. Subunit d1 and d2 were purified as homogeneous monomers as demonstrated by dynamic light scattering (DLS) analysis. Secondary structures of d subunits were estimated to be composed of 73% α-helix and 2% β-sheet, as analyzed using circular dichroism (CD) analysis. Although sequence identity and secondary structures of d subunits were highly similar, the relative stability against thermal stress was higher for d1 than d2. Efficient expression and purification of d subunits, together with biophysical and biochemical characterization, presented in this study is expected to facilitate further structural analysis to clarify specific inter-molecular interactions involved in multi-subunit assembly and regulation of H(+) transporters.
authors.affiliation: Division of Electron Microscopic Research, Korea Basic Science Institute (KBSI), 113 Gwahangno, Yuseong-gu, Daejeon 305-333, Republic of Korea; Graduate School of Analytical Science and Technology (GRAST), Chungnam National University, Daejeon 305-764, Republic of Korea.
authors.forename: Hyosun
authors.initials: H
authors.lastname: Lim
authors.affiliation: Division of Magnetic Resonance, Korea Basic Science Institute (KBSI), 16 Yeongudanji-Ro, Ochang, Chungbuk 363-883, Republic of Korea.
authors.forename: Hae-Kap
authors.initials: HK
authors.lastname: Cheong
authors.affiliation: Department of Bioscience & Biotechnology, Chungnam National University, Daejeon 305-764, Republic of Korea; Graduate School of Analytical Science and Technology (GRAST), Chungnam National University, Daejeon 305-764, Republic of Korea.
authors.forename: Jae-Rang
authors.initials: JR
authors.lastname: Rho
authors.affiliation: Division of Electron Microscopic Research, Korea Basic Science Institute (KBSI), 113 Gwahangno, Yuseong-gu, Daejeon 305-333, Republic of Korea. Electronic address: hjk002@kbsi.re.kr.
authors.forename: Jae-Kyung
authors.initials: JK
authors.lastname: Hyun
authors.affiliation: Division of Electron Microscopic Research, Korea Basic Science Institute (KBSI), 113 Gwahangno, Yuseong-gu, Daejeon 305-333, Republic of Korea; Graduate School of Analytical Science and Technology (GRAST), Chungnam National University, Daejeon 305-764, Republic of Korea. Electronic address: y-jkim@kbsi.re.kr.
authors.forename: Youn-Joong
authors.initials: YJ
authors.lastname: Kim
mesh_terms: D000595:Amino Acid Sequence; D002942:Circular Dichroism; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D015870:Gene Expression; D006801:Humans; D008969:Molecular Sequence Data; D055550:Protein Stability; D017433:Protein Structure, Secondary; D021122:Protein Subunits / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D016415:Sequence Alignment; D025262:Vacuolar Proton-Translocating ATPases / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism
chemical_list: D021122:Protein Subunits; D025262:Vacuolar Proton-Translocating ATPases
keywords: Protein over-expression; Thermal stability; V-ATPase subunit d1; V-ATPase subunit d2