Artificial Synthesis and Cloning of the Sweet-tasting Protein Brazzein Coding Gene

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.