Author(s):
Aguiar, Tatiana Quinta ; Leal, Tânia ; Rodrigues, Diana G. ; Abrunhosa, Luís ; Oliveira, Carla ; Domingues, Lucília
Date: 2025
Persistent ID: https://hdl.handle.net/1822/93579
Origin: RepositóriUM - Universidade do Minho
Project/scholarship:
info:eu-repo/grantAgreement/FCT/6817 - DCRRNI ID/UIDB%2F04469%2F2020/PT;
info:eu-repo/grantAgreement/FCT/CEEC IND 2017/CEECIND%2F00728%2F2017%2FCP1458%2FCT0004/PT;
Subject(s): Ochratoxin A; BSA domain II; Bioreceptor; Solid-phase extraction; Recombinant protein
Description
Available online 30 October 2024
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/Lculture 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.
This study was supported by the Portuguese Foundation for Science and Technology (FCT) under the scope of the strategic funding of UIDB/04469/2020 unit https://doi.org/10.54499/UIDB/04469/2020) and Project MycoProAffinity (https://doi.org/10.54499/2022.03438. PTDC). Luís Abrunhosa acknowledges FCT for the assistant research contract CEECIND/00728/2017. The authors would also like to acknowledge Heiðar Aðalsteinsson, Lavínia Pinto and Rui M. Rodrigues for their assistance in CD spectroscopy analyses.
info:eu-repo/semantics/publishedVersion
