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

Summary

• 1. Calcium binding properties were examined in VAT-1, an abundant 41-kDa membrane protein expressed in the cholinergic cynaptic vesicles ofTorpedo.

• 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 glutathioneS-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 130µM.

• 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.