VAT-1 from Torpedo synaptic vesicles is a calcium binding protein: a study in bacterial expression systems

Orit Levius, Michal Linial*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

8 Scopus citations

Abstract

1. Calcium binding properties were examined in VAT-1, an abundant 41-kDa membrane protein expressed in the cholinergic cynaptic vesicles of Torpedo. 2. An overlay assay, using45Ca2+ as a tracer, demonstrated the ability of a recombinant VAT-1 produced from the IPTG-inducible pKK223-3 expression vector to bind calcium. 3. A high yield of recombinant VAT-1 was obtained from the glutathione S-transferase (GST) expression system. The fusion product enabled VAT-1 purification via affinity chromatography. Subsequent cleavage by thrombin resulted in its separation from the GST carrier protein. 4. A direct Ca2+-binding study was performed with purified VAT-1 by a quick-spin column technique, in the presence of45Ca2+. Quantitative analysis revealed a 1:1 molar stoichiometry for binding of Ca2+ to VAT-1, with a dissociation constant of 130 μM. 5. A GST-linked truncated protein consisting of 13 kDa from the VAT-1 carboxy-terminal domain was found to retain the capacity to bind Ca2+. 6. A data search for homologies between VAT-1 and known Ca2+-binding proetins revealed considerable similarity to members of the annexin family in a 140-amino acid region from the carboxy terminal of VAT-1, which overlaps two tandem Ca2+-binding domains of the annexin proteins.

Original languageAmerican English
Pages (from-to)483-492
Number of pages10
JournalCellular and Molecular Neurobiology
Volume13
Issue number5
DOIs
StatePublished - Oct 1993

Keywords

  • Torpedo
  • annexin
  • bacterial expression
  • calcium binding protein
  • electric organ
  • synaptic vesicles

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