Cloning, purification, and nucleotide-binding traits of the catalytic subunit A of the V1VO ATPase from Aedes albopictus

Cornelia Hunke, Wei June Chen, Hans Jochen Schäfer, Gerhard Grüber*

*Corresponding author for this work

Research output: Contribution to journalJournal Article peer-review

20 Scopus citations


The Asian tiger mosquito, Aedes albopictus, is commonly infected by the gregarine parasite Ascogregarina taiwanensis, which develops extracellularly in the midgut of infected larvae. The intracellular trophozoites are usually confined within a parasitophorous vacuole, whose acidification is generated and controlled by the V1VO ATPase. This proton pump is driven by ATP hydrolysis, catalyzed inside the major subunit A. The subunit A encoding gene of the Aedes albopictus V1VO ATPase was cloned in pET9d1-His3 and the recombinant protein, expressed in the Escherichia coli Rosetta™ 2 (DE3) strain, purified by immobilized metal affinity- and ion-exchange chromatography. The purified protein was soluble and properly folded. Analysis of secondary structure by circular dichroism spectroscopy showed that subunit A comprises 43% α-helix, 25% β-sheet and 40% random coil content. The ability of subunit A of eukaryotic V-ATPases to bind ATP and/or ADP is demonstrated by photoaffinity labeling and fluorescence correlation spectroscopy (FCS). Quantitation of the FCS data indicates that the ADP-analogues bind slightly weaker to subunit A than the ATP-analogues. Tryptophan fluorescence quenching of subunit A after binding of different nucleotides provides evidence for secondary structural alterations in this subunit caused by nucleotide-binding.

Original languageEnglish
Pages (from-to)378-383
Number of pages6
JournalProtein Expression and Purification
Issue number2
StatePublished - 06 2007


  • Fluorescence correlation spectroscopy
  • Mosquito
  • Photoaffinity labeling
  • Subunit A
  • V ATPase
  • VV ATPase
  • Vacuolar-type ATPase


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