Today, sucrose intake caused problems for people’s health. So, the demand for low-caloric natural sweeteners with good taste properties such as sweet proteins increased. Among the sweeteners, Brazzein is an attractive alternative to sucrose. Because of its intense sweetness, natural origin and good stability. Brazzein was isolated from the fruit of the African plant Pentadiplandra hrazzeana Baillon, that is impractical to produce economically on a large scale. Therefore, widespread commercial production of this protein will probably require the transfer of protein expression to a heterologous system by means of recombinant DNA technology. In this study, due to the unavailability of genome of P. brazzeana Baillon plant for Brazzein gene isolation, artificial synthesis of Brazzein gene was done by the assembly of PCR and SOEing PCR. Amino acid sequence of Brazzein protein was translated to 162 nucleotides based on the preferred codon usage of maize using the Emboss Backtranseq program. Then, by using six overlapping primers in five consecutive PCR reactions Brazzein gene sequence was synthesized. The results of the PCR reaction showed accuracy. Then the synthetic fragment was cloned into pBI121 plant expression vectors that contained a constitutive promoter CaMV35S and seed-specific promoter Napin, which was confirmed by nucleotide sequencing.

A sweet water-soluble protein that reminds stable over wide ranges of temperature and pH, Brazzein has various applications. Its tastes like cane sugar but have no calories. However, the extraction of brazzein from its natural source is expensive and not applicable. In this study we used recombinant DNA technology to provide an alternative option for cheaper mass production of brazzein. A brazzein gene was designed, synthesized by using oligonucleotids and cloned into the pBlueScript vector. The synthetic fragment linked to the C-terminal of Maltose-binding protein (MBP) and Glutathion-S-Transferase (GST) for protein expression. The recombinant protein was expressed as a soluble form after induction by IPTG. The fusion protein was purified by amylose resin column and detected by SDS-PAGE. The best yields were achieved by producing brazzein as a fusion with MBP. MBP-brazzein system permits large-scale functional expression and purification of recombinant soluble proteins, providing a basis for the future study of structure and function of brazzein.