Otimização do protocolo de expressão heteróloga da Frutalina recombinante (rFTL) ativa em Komagataella phaffii

Abstract

Frutalin (FTL) is a plant lectin extracted from the seeds of Artocarpus incisa that belongs to the Jacalin-related lectin family, exhibiting affinity for α-D-galactose residues and several biological applications described in the literature. However, FTL naturally occurs as a complex mixture of multiple glycosylated isoforms, and limitations associated with protein extraction from plant sources hinder its large-scale application, making heterologous expression a promising alternative for obtaining a more uniform product. In this context, the present study aimed to optimize the heterologous expression of Frutalin in Komagataella phaffii to obtain an active recombinant protein with a high degree of purity and preserved carbohydrate-binding activity. Protein expression was carried out under different combinations of temperature (15°C and 30°C) and methanol inducer concentration (0.5% and 1.0%), and was monitored by Bradford assay, SDS-PAGE, Dot Blot, and Western Blot using anti-FTL antibodies. The most promising expression condition was selected for purification by immobilized metal affinity chromatography (IMAC), followed by dialysis and concentration. Subsequently, the purified protein was characterized by dynamic light scattering (DLS), both individually and in association with human secretory immunoglobulin A1 (sIgA1). The results demonstrated that the expression condition combining 30°C and 0.5% methanol provided the highest total protein recovery, reaching approximately 654 µg/mL in the ammonium sulfate-precipitated fraction. The presence of recombinant Frutalin (rFTL) was confirmed by SDS-PAGE and Western Blot analyses, which revealed immunoreactive bands between 20 and 25 kDa. The purified recombinant protein was further analyzed by DLS, which revealed hydrodynamic size distributions consistent with the formation of supramolecular rFTL complexes, as evidenced by an increase in the average hydrodynamic diameter and a reduction in the polydispersity indices of the complexes compared with the individual controls. Overall, the obtained results suggest that the recombinant protein retained its lectin activity, reinforcing its potential for future biotechnological application.