HETEROLOGOUS EXPRESSION AND PURIFICATION OF RECOMBINANT VARIANTS OF THE PISUM SATIVUM DEFENSIN PSD1

Fungal infections represent a growing public health concern, particularly due to the emergence of antifungal resistance and the limited number of available therapeutic drug classes. Plant defensins are promising candidates for the development of alternative antifungal agents. Psd1, a 5.4 kDa defensin from Pisum sativum, exhibits antifungal activity and low toxicity toward human cells, attributed to its selective interactions with fungal membrane components, including ergosterol and glucosylceramide. Molecular dynamics simulations performed by our group identified three Psd1 variants, m374 (V13W, T16W, N17W, D21R), m1344 (D8R, V13W, T16W, N17W), and m2318 (D8W, V13W, T16W, N17W, D21R), which showed increased membrane binding and penetration compared with the native protein.This study aimed to establish an optimized workflow for the heterologous expression and purification of these variants for subsequent structural and functional characterization. Plasmids encoding native Psd1 and its variants were amplified in Escherichia coli DH5α, purified, and validated by Sanger sequencing. Sequence alignment confirmed plasmid identity and the presence of the expected mutations. The plasmids were linearized with SalI and used to transform Komagataella phaffii. Recombinant expression was performed in BMG medium for 120 h at 28 °C, with daily supplementation of 0.7% methanol. SDS-PAGE analysis detected bands compatible with the expected molecular mass of Psd1 (~5.4 kDa) from 24 h onward, with increasing intensity throughout induction, indicating progressive accumulation of recombinant peptides in the culture supernatant. Culture supernatants were subjected to size-exclusion chromatography using Sephacryl S-100, followed by SDS-PAGE analysis of the collected fractions. The native protein displayed an elution profile consistent with the expected molecular mass, with its main peak eluting at approximately 220 mL. In contrast, the variants exhibited an additional peak at approximately 100 mL, consistent with an apparent higher molecular mass. This profile may reflect oligomerization under the chromatographic conditions, potentially associated with their increased tryptophan content. Selected fractions will be further purified by reversed-phase chromatography on a C8 column using an acetonitrile gradient Overall, these results establish an optimized pipeline for the recombinant production in K. phaffii and sequential purification of Psd1 variants providing a basis for their subsequent structural and functional characterization.