Author(s): Rodrigues, Diana Isabel Gomes ; Adalsteinsson, Heidar Már ; Pinto, Lavínia ; Aguiar, Tatiana Quinta ; Oliveira, Carla ; Abrunhosa, Luís ; Domingues, Lucília
Date: 2023
Persistent ID: https://hdl.handle.net/1822/88264
Origin: RepositóriUM - Universidade do Minho
Project/scholarship: info:eu-repo/grantAgreement/FCT/6817 - DCRRNI ID/UIDB%2F04469%2F2020/PT;
Subject(s): Human serum albumin; Pichia pastoris; Recombinant production; Mycotoxin binding
Human serum albumin (HSA) is a monomeric multidomain macromolecule with wide clinical and biotechnological applications. Thus, there is a high demand for HSA, which is increasing due to new emerging applications. Among these, the extraordinary ligand binding capacity of HSA has been recently explored to capture mycotoxins from food and feed matrices. However, HSA is currently produced commercially primarily from human plasma, which conveys supply limitations. Recombinant production may be a solution to overcome this obstacle. Among the expression systems explored for HSA production, the yeast Pichia pastoris has drawn attention for its simplicity of genetic manipulation and high-cell density in low-priced medium, along with efficient secretory capacity and ability to form disulfide bonds, which are major advantages over bacterial systems. Envisioning cost-effective production of recombinant HSA for its widespread use in mycotoxin capture devices, P. pastoris KM71H was transformed with the pPICZ9ssHSAH6 vector to produce recombinant HSA in fusion with the His6 tag at the C-terminal (rHSA). After selecting the bestperforming clones, minimal and complex media were tested for rHSA production in shake-flask. The best production of stable extracellular rHSA was achieved in complex medium within 96 h of methanol induction at nearly 0.5 g/L culture. Following rHSA purification by immobilized metal ion affinity chromatography, fluorescence quenching studies were used to assess the suitability of the produced rHSA for the capture of different mycotoxins. The binding constants (Ksv, L/mol) obtained for rHSA and commercial HSA were: 8.29x105 and 6.22x105 for ochratoxin A, 6.02x104 and 2.97x104 for zearalenone, and 1.26x104 and 9.50x103 for patulin, respectively. rHSA thus displayed results that matched those of native HSA, forming strong complexes with these mycotoxins.
This work was supported by the Portuguese Foundation for Science and Technology (FCT) under the scope of the strategic funding of UIDB/04469/2020 unit and project MycoProAffinity 2022.03438.PTDC (https://doi.org/10.54499/2022.03438.PTDC)
info:eu-repo/semantics/publishedVersion
