Expression Of Sweet Protein Brazzein In The Methylotrophic Yeast Pichia Pastoris

Brazzein is a small, heat-stable, intensely sweet protein first isolated from the fruit of Pentadiplandra brazzeana Baillon found in West Africa in 1994. It is 2,000 times sweeter than sucrose in comparison to 2% sucrose aqueous solution .Because of its intense sweetness and stability at high pH and temperature, brazzein is an ideal system for investigating the chemical and structural requirements involved in sweet-taste properties. Pichia pastoris is a newly-developed hererologous gene expression system has been utilized to produce attractive levels of a variety of intracellular and extracellular protein of interest. It can express heterologous soluble protein and secret out of cell . OBJECTIVE: In this study, we attempted to clone brazzein gene into methanol nutritional vector-pPIC9K alpha-A, linked with alpha-factor signal peptide. Then transforme recombinant vector into Pichia pastoris yeast strain GS115. At last produce a recombinant brazzein having biological activity. METHODS: According to the brazzein gene sequence and Pichia pastoris preference codon uasage, the brazzein gene was designed and synthesized. BamHI+EcoRI cutting sites were introduced at both the N-and C-terminal ends by genetic modification to convenience of clone. We add the 5'AOX1 primer and 3'AOX1 primer amplify pPIC9K and alpha-factor signal peptide sequence was obtained.T4DNA ligase ligate pPUC19 and alpha-factor signal peptide sequence, which were cut BamHI+EcoRI. Recombinant vector pPUC19S was gotten Brazzein cut with BamHI+EcoRI was cloned in the a pPIC9K vector. Recombinant vector pPIC9KB was gotten. .BglII restriction enzyme pPIC9KB,then electroporation transformed into Pichia pastoris yeast strain GS115. Recombinant brazzein was expressed and secreted by the methylotrophic yeast. SDS-PAGE analyze that recombinant protein is secreted at approximately 150mg/L of culture supernatant and yielded an expression level of over 80% of total cellular secreted protein. Monomer displays a molecular weight of 6.5kDa. CONCLUSION: brazzein gene was cloned into yeast cell and its expressive products has high activity of sweet-taste. Recombinant brazzein is secreted at approximately 150mg/L of culture supernatant and yielded an expression level of over 80% of total cellular secreted protein.