Molecular cloning, expression, and characterization of a major 38-kd cochineal allergen

Background

Carmine is a natural red pigment obtained from dried gravid female cochineal insects (Dactylopius coccus or Coccus cacti). There have been several reports of allergies to carmine, but the major allergens responsible have not been identified.

Objective

To identify the major allergenic proteins in cochineal.

Methods

Immunoblots of purified cochineal extract were probed with sera from 3 patients with allergy. Partial amino acid sequences were determined for the proteins bound by IgE, and the corresponding cDNA, containing a complete coding region, was cloned by 5′ and 3′ rapid cDNA extension and PCR. The recombinant protein was expressed in yeast and subjected to immunoblotting.

Results

We identified a full-length cDNA encoding a protein, which we named CC38K, with 335 amino acids and a molecular mass calculated as 38 kd. This amino acid sequence included all the partial amino acid sequences obtained from the purified proteins identified by IgE from patients with allergy. Recombinant CC38K protein was recognized by patients' sera, indicating that this is a major allergen present in carmine. The CC38K sequence showed homology to phospholipases.

Conclusion

We have, for the first time, identified the major allergen in cochineal extract. This protein may be a phospholipase or related enzyme, both of which are known to be allergens in other insects.

Key words: Cochineal, allergen, cDNA, IgE, food allergy, carmine, immunoblotting, cloning, recombinant

Abbreviations used: C1, Whole coding region of CC38K cDNA, C22, Truncated CC38K cDNA encoding the processed form, CC38K, Major allergenic protein (38 kd) in cochineal extracts, 38K cDNA, Full-length cDNA of major allergenic protein (38 kd) in cochineal extracts, PLA₁, Phospholipase A₁, RACE, Rapid amplification of cDNA ends