Heterologous Production and Characterization of Rapeseed Procruciferin Isoforms for Sustainable Food Protein Development

ABSTRACT Cruciferins, the major seed storage proteins in rapeseeds ( Brassica napus ), are promising candidates for sustainable food protein sources due to their well‐balanced amino acid composition. However, their functional properties remain poorly understood, particularly at the individual isoform level. In this study, the recombinant production of six cruciferin isoforms (Cru‐1, Cru‐2, Cru‐3, Cru‐4, Cru‐A, and Cru‐2/3A) was systematically explored in Escherichia coli using 13 different expression strains and three vectors. This resulted in three of the isoforms, Cru‐1, Cru‐A, and Cru‐2/3A, being successfully obtained in soluble form, purified to homogeneity in yields of 1.5–4.0 mg/g cells, and characterized by differential scanning fluorimetry (DSF), mass photometry, circular dichroism spectroscopy, as well as matrix‐assisted laser desorption/ionization–time of flight mass spectrometry. All three isoforms appeared as their unprocessed single polypeptide chain proproteins, complying with the experimental and theoretical molecular mass, and formed trimeric complexes. Moreover, they exhibited cooperative thermal unfolding transitions and were stabilized by disulfide bonds as monitored by DSF in the absence and presence of dithiothreitol. Higher salt concentration and neutral‐to‐alkaline pH improved the aggregation resistance during thermal unfolding. Proteolytic cleavage of Cru‐1 using proteinase K raised the thermal denaturation temperature and promoted hexamer formation, mimicking the native oligomeric state of cruciferin observed in seeds. These insights into isoform‐specific thermal stability and assembly may inform the design of plant‐based food ingredients with tailored textural and thermal properties.