RECOMBINANT CHENOPODIN: QUATERNARY ASSEMBLY AND UNPLANNED CLEAVEG CHALLENGES

The demand for sustainable, plant-based protein sources has drawn increasing attention to quinoa (Chenopodium quinoa) while growing interest in plant-derived bioactive proteins has raised the need for efficient and sustainable production strategies [1,2]. Among quinoa storage proteins, Chenopodins—hetero-hexameric globulins composed of six mature oligomers—are of particular interest, with the HcC isoform previously shown to exert immunomodulatory effects on Caco-2 cells [3]. As with other plant globulins, Chenopodin maturation involves proteolytic cleavage separating the precursor into α and β subunits linked by a disulfide bond; however, the enzymes and mechanisms underlying this process remain poorly characterized. Recombinant protein expression represent an interesting alternative to extraction, assuring scalable production and reducing environmental impact. However, obtaining high amounts of pure, native protein is crucial to produce the anti-inflammatory protein and to validate the molecular mechanism of its bioactivity. This work addresses a central challenge in recombinant plant protein production: reconstituting the native hexameric oligomerization state in vitro, a prerequisite for a protein that is structurally and functionally representative of its natural counterpart. To this purpose we introduced a Factor Xa cleavage site between α and β subunits to simulate natural post-translational cleavage and promote quaternary assembly. Preliminary results confirm the feasibility of Chenopodin overexpression, however Factor Xa provided some off-target cleavages, yielding differently processed HcCs alongside the expected mature protein. We investigated the cause of these results through bidimensional electrophoresis combined with sequence and structure analysis, to plan further optimization steps. Results show that the major off-target cleavage involves a surface-exposed GR sequence, yielding a short HcC where the β subunit remains bound via the conventional S-S bridge to a shorter α subunit. These results open the possibility of modifying the GR sequence by SDM to avoid undesired cleavage and obtain a mature, correctly oligomerized protein resembling the natural quinoa counterpart. References (max 4): [1] Scanlin L, Lewis KA. Quinoa as a sustainable protein source: production, nutrition, and processing. In: Sustainable Protein Sources. Elsevier. 2017: 223–238. [2] Náthia-Neves G, Getachew AT, Santana ÁL, Jacobsen C. Legume proteins in food products: extraction techniques, functional properties, and current challenges. Foods. 2025; 14: 1626. [3] Capraro J, De Benedetti S, Di Dio M, Bona E, Abate A, Corsetto PA, Scarafoni A. Characterization of chenopodin isoforms from quinoa seeds and assessment of their potential anti-inflammatory activity in Caco-2 cells. Biomolecules. 2020; 10: 795.