Recombinant bovine serum albumin domain II as bioreceptor for ochratoxin A capture

Established chromatographic techniques for mycotoxin control in foodstuffs require prior sample enrichment and clean-up, typically achieved using immunoaffinity columns (IACs). Bovine serum albumin (BSA) has recently emerged as a cost-effective alternative to antibodies used in IACs. This study aimed at exploring the BSA domain II (BDII), which houses the primary binding site for ochratoxin A (OTA), as a bioreceptor for OTA capture. Recombinant BDII (rBDII) was produced in soluble form by Escherichia coli Origami 2(DE3), fused to a His6 (HisBDII) or thioredoxin-His6 (TrxBDII) tag, with yields up to 19 ± 4.3 mg/L_culture in shake-flask. Fluorescence and circular dichroism (CD) spectroscopy revealed interaction of OTA with both rBDII variants, with estimated binding constants for OTA-HisBDII/TrxBDII complexes in the range of 5.7-9.3 × 10⁴ M^-1. CD also showed an α/β structure of rBDII variants, in opposition to the predominant α-helical structure of whole BSA, and slight increase in their α-helical content upon binding to OTA. TrxBDII immobilized on Ni-NTA resin successfully captured OTA from spiked samples at the optimum pH range of 6.5-7.0, allowing OTA extraction, clean-up, and enrichment from spiked white grape juice, with up to 84 ± 7.4 % recovery.