pBI121 vector

Brazzein - a low-calorie natural sweetener that is an effective alternative to the harmful effects of sucrose. To date, limitations in the production of recombinant brazzein proteins using heterologous expression systems have been reported, as well as challenges with solubility and low yields. Anticipating the potential of brazzein genes in therapeutics, molecular strategies are needed to optimize the production of recombinant brazzein using different prokaryotic and eukaryotic expression systems. In this study, the brazzein gene was first cloned into the plant expression vector pBI121 with the CaMV35S promoter region. The heat shock or calcium chloride method was optimized for transformation into E. coli (DH5-α) using the recombinant vector pBI121. Kanamycin resistance selection, colony PCR, and transformation efficiency analysis were performed to analyze the successful cloning process. Studies have shown that the cloned brazzien gene in the PbI121 vector (plant expression vector) can provide a heterologous expression system as it can be used to express recombinant brazzien proteins in various plant species regardless of their native plant.

In this study, transformed cultures with brazzein gene cloned in pBI121 vector with CaMV35S promoter can be used to transform different plants such as sorghum, sugarcane, and radish to enhance sweetness and may provide a platform for commercial scale production of therapeutic proteins using crop plants as bioreactor. This transformation cassette can be used to develop transgenic plants with improved quality and taste. It would also remove geographic barriers to developing and producing transgenics of this valuable protein on a commercial scale.

Figure 1. In the process of cloning the brazzein gene, the digested brazzein gene from BamHI and SacI was inserted into the digested pBI121ـPro-Adh:TaPCS1:cmyc plasmid with the same restriction enzymes to prepare the pBI121-brz plasmid. E. coli was transformed with the ligation plasmid in the transformation of cloned sections, which was confirmed by antibiotic selection and colony PCR analysis. (Saleh B, et al. 2023)