Desenvolvimento de uma linhagem de Komagataella phaffii para produção recombinante da Pumpkin Phloem Lectin (PPL) de Cucurbita máxima

Abstract

Lectins are proteins that have the ability to bind to carbohydrates in a specific and reversible manner, making them applicable in various fields. The Pumpkin Phloem Lectin (PPL) has specificity for chito-oligosaccharides and RNA and is associated with the plant's defense mechanisms against pests and pathogens. However, obtaining the protein from the phloem using classical purification methods does not yield high quantities, which may limit future biotechnological applications. Therefore, the objective of this work was to obtain a genetically modified Komagataella phaffii strain for the recombinant expression of Pumpkin Phloem Lectin (PPL) from Cucurbita maxima. For the design of the expression vector (SnapGene 6.1), the gene sequence of PPL was collected from Uniprot and optimized for K. phaffii (Genscript). The sequence was synthesized by Genone in the pPICZαA vector (Invitrogen). Escherichia coli TOP 10 strains were transformed for replication, followed by extraction and linearization of the DNA with Sac I. Competent cells of K. phaffii X-33 were prepared and transformed by electroporation. The analysis of the transformants was performed by PCR and agarose gel electrophoresis. Colonies were selected for zeocin resistance testing (100-2000 μg/mL). Expression was carried out in BMMY medium with methanol (1% and 2%) and, in a second test, with sorbitol. The supernatants were precipitated with ammonium sulfate (0-80%), dialyzed, and concentrated. Expression was analyzed by hemagglutinating activity and SDSPAGE. After genetic transformation, isolated colonies of K. phaffii X-33 were observed at the end of the second day of incubation. Colony PCR showed bands of 1150 bp, corresponding to the AOX gene. In the zeocin resistance test, all colonies grew at the highest concentrations. SDS-PAGE analyses indicated bands at the expected molecular weight for PPL, but the hemagglutinating activity assay was inconclusive, as proteins from the untransformed culture also showed activity. Thus, it is concluded that the genetic transformation of K. phaffii was successful and that the obtained colonies are suitable for expression testing. SDS-PAGE analysis suggested the presence of PPL, but without definitive confirmation. More specific methods, such as Western blot and mass spectrometry, along with optimization of cultivation conditions, are alternatives to validate PPL expression and enable its biotechnological application.