利用毕赤酵母表达植物甜蛋白(brazzein)的初步研究

Secret expression of plant sweet-tasting protein brazzein in yeast pichia pastoris

实验将含有Brazzein基因的pPIC9K穿梭质粒通过电导入巴斯德比赤酵母(Pichiapastori)GS115中,并从22个阳性克隆中筛选出His+Muts高拷贝转化子2个,经诱导表达,产物进行Tricine SDS PAGE电泳鉴定,目标蛋白的相对分子量与理论值一致,又经小试(5～10L罐)蛋白产量可达385mg/L。利用大孔树脂D152初步分离,得到有微甜味的产物。进一步纯化后获得了1D NMR图谱。

Brazzein is a natural sweet tasting protein firstly extracted from a high plant distributing in west Africa. Brazzein possesses potential commercial value due to its characterization of high sweetness and high heating stability. Some primary research results were obtained in our lab. A synthesis DNA fragment coding for brazzein was inserted into the plasmid pPIC9K to construct the expression vector pPIC9K-B which was transformed into the host cell yeast Pichia pastoris GS115 by means of eletroporation. By a series of screening 22 positive clones were identified. Among them 2 clones bear a high copy of the purpose gene. The behavior of the target protein on Tricine-SDS-PAGE was identifiable to its molecular weight 6473. Moreover, one clone was further investigated by fermentation in a small scale and its raw product with sweet taste was 385?mg/L.