Expression and characterization of the antifungal protein PtAFP from Pyrenophora tritci-repentis by synonymous codon bias in Escherichia coli

The exploration of defense peptides holds significant research potential for fungal inhibition. This study optimized the antifungal gene (PtAFP) from Pyrenophora tritici-repentis using synonymous codon bias. The resulting antifungal protein PtAFP exhibited a characteristic antiparallel β-folded barrel structure, featuring three disulfide bonds, with the initial 23 amino acids forming a signal secretion sequence. Successful cloning of the optimized PtAFP gene into Escherichia coli yielded an antifungal protein of approximately 10 kDa. Fluorescence analysis assessing the thermal stability of PtAFP revealed a remarkable retention of 87% fluorescence intensity after 6 h at 50°C, underscoring its robust protein stability. Inhibition rate analysis against Penicillium funiculosum CICC40279 indicated a 15% inhibition rate after 48 h of cultivation, persisting at 18% even after 72 h. This study highlights the effective application of the optimized gene sequence in E. coli, laying the groundwork for future advancements in the development of more potent antifungal proteins.