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

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.