Lectins are glycan-binding proteins involved in diverse biological processes and have gained attention for their potential applications in biotechnology and immunomodulation. BOL (Brassica oleracea lectin) is a unique ~34 kDa lectin isolated from Brassica oleracea var. botrytis, composed exclusively of TRAF-like domains, where TRAF stands for tumor necrosis factor receptor–associated factor. To overcome the limitations of plant-based extraction, we aimed to produce recombinant BOL in Escherichia coli. Various strains and expression vectors were tested under distinct induction conditions to optimize solubility and yield. While expression using pET28a was unsuccessful, GST-tagged BOL was efficiently expressed in E. coli BL21-R3-pRARE2(DE3) and purified using affinity chromatography. Functional assays demonstrated that the recombinant protein retained lectin activity, as evidenced by hemagglutination of goat erythrocytes. Protein identity was confirmed by MALDI-TOF/TOF mass spectrometry, with tryptic peptides matching the BOL lectin sequence in the National Center for Biotechnology Information (NCBI) database. Our findings highlight the importance of codon optimization, temperature modulation, and fusion tag selection for the successful expression of eukaryotic lectins in E. coli. This work provides a platform for future functional studies of BOL and supports its potential application in plant immunity and biomedical research.

The BOL lectin, a 34 kDa protein with a hemagglutination titer of 64 hemagglutination units (HU), was extracted from cauliflower (Brassica oleracea spp. botrytis L.), purified by affinity and ion exchange chromatography, and confirmed, in this study, by Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE). The antibiofilm activity of BOL was evaluated at two concentrations (0.1 mg/mL and 1.0 mg/mL) against bacterial strains of importance to human health (Bacillus cereus ATCC 10876, Escherichia coli ATCC 25922, Staphylococcus aureus ATCC 29213, and Streptococcus agalactiae ATCC 12403). In addition to a biofilm formation assay, a pre-formed biofilm assay was conducted, with biofilm structure analyzed by Scanning Electron Microscopy (SEM). The antimicrobial potential of BOL was also investigated using the Minimum Inhibitory Concentration (MIC) assay in 96-well microplates. Among the tested bacterial strains, BOL exhibited activity against S. aureus at 1.0 mg/mL, interfering with both biofilm formation and disrupting pre-formed biofilms, which may be explained by a possible interaction between BOL and the components present in the biofilm matrix. However, no antibiofilm activity was observed against E. coli, B. cereus, or S. agalactiae, possibly due to differences in the composition of their biofilm matrices. Furthermore, BOL showed no detectable bactericidal or bacteriostatic activity in the antimicrobial assays. In conclusion, BOL lectin, at the tested concentrations, does not exhibit direct antimicrobial activity but effectively disrupts the extracellular matrix in S. aureus ATCC 29213.