Establishment Of Brazzein Expression System In Yeast

Brazzein is a kind of natural sweet-taste protein which has the characteristics of intensely sweetness,low grade-fever,heat-stable,asepsis and multifunction. It was 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. But the natural resources of Brazzein was limited, while the cost of isolation is too expensive, In addition, the study on Brazzein with genetic engineering has many problems. Such as in the prokaryotic expression system, it is difficult to isolate the sweet protein from cytorrhyctes, and also have the limitation of low quality and non sweetness of recombinant protein. While the article about the expression in pichia yeast haven't been reported yet.METHODS: According to the sequence of brazzein gene and Pichia pastoris preference codon usage, the fist amio acid of Brazzein was knockouted, the Asp29 and Glu41 were changed into Asn and Lys respectively, and four pairs of primers were designed and synthesized. Xho I+XbaI cutting sites were introduced at both the N-and C-terminal ends by genetic modification to convenience of clone. Finally the Brazzein gene was amplified by SOE-PCR, total length of the product is 188bp.After the identification of clony-PCR and enzyme digestion, successfully constructed the clone plasmid pUC57- Bra. The Brazzein fragment, produced from pUC57- Bra by Xho I and Xba I was ligated with the yeast expressing vector pGAPZαA which was also digested by Xho I and Xba I, then the ligation was transformed to E.coli. The results of PCR and enzyme digestion of plasmid proved that recombination vector was obtained (named SMD1168/pGAPZαA-Bra).After linealization of the plasmids with AvrⅡ,Brazzein recombination gene was inserted into the downstream of high performance promoter AOXI, integrated into the genome of host yeast Pichia Pastoris SMD1168 by electroporation. After the optimization of expression conditions the recombination protein was expressed successfully in Pichia yeast. SDS-PAGE analyzing indicated a molecular weight of 6.5kDa protein was obtained at inducing 72 hours .The expression quantity of the recombinant protein amounted to 0.2g/L.The result of sugariness determination on purified protein by tasting showed that Brazzein recombinant protein has high biological activity.CONCLUSION: In this study, we cloned brazzein gene into eukaryotic expression vector pGAPZαA, constructed the recombinant vector pGAPZαA-Bra. Then transformed it into Pichia pastoris yeast strain SMD1168.At last produced a recombinant brazzein protein having biological activity.