Ide Nobuyuki, Masuda Tetsuya, Kitabatake Naofumi
Division of Food Science and Biotechnology, Graduate School of Agriculture, Kyoto University, Uji, Kyoto 611-0011, Japan.
Biochem Biophys Res Commun. 2007 Nov 23;363(3):708-14. doi: 10.1016/j.bbrc.2007.09.021. Epub 2007 Sep 18.
Thaumatin is a 22-kDa sweet-tasting protein containing eight disulfide bonds. When thaumatin is expressed in Pichia pastoris using the thaumatin cDNA fused with both the alpha-factor signal sequence and the Kex2 protease cleavage site from Saccharomyces cerevisiae, the N-terminal sequence of the secreted thaumatin molecule is not processed correctly. To examine the role of the thaumatin cDNA-encoded N-terminal pre-sequence and C-terminal pro-sequence on the processing of thaumatin and efficiency of thaumatin production in P. pastoris, four expression plasmids with different pre-sequence and pro-sequence were constructed and transformed into P. pastoris. The transformants containing pre-thaumatin gene that has the native plant signal, secreted thaumatin molecules in the medium. The N-terminal amino acid sequence of the secreted thaumatin molecule was processed correctly. The production yield of thaumatin was not affected by the C-terminal pro-sequence, and the pro-sequence was not processed in P. pastoris, indicating that pro-sequence is not necessary for thaumatin synthesis.
奇异果甜蛋白是一种含有八个二硫键的22千道尔顿甜味蛋白。当使用与来自酿酒酵母的α-因子信号序列和Kex2蛋白酶切割位点融合的奇异果甜蛋白cDNA在毕赤酵母中表达奇异果甜蛋白时，分泌的奇异果甜蛋白分子的N端序列未被正确加工。为了研究奇异果甜蛋白cDNA编码的N端前序列和C端前序列在奇异果甜蛋白加工及毕赤酵母中奇异果甜蛋白生产效率方面的作用，构建了四种具有不同前序列和前序列的表达质粒，并将其转化到毕赤酵母中。含有具有天然植物信号的前奇异果甜蛋白基因的转化体在培养基中分泌奇异果甜蛋白分子。分泌的奇异果甜蛋白分子的N端氨基酸序列被正确加工。奇异果甜蛋白的产量不受C端前序列的影响，且前序列在毕赤酵母中未被加工，这表明前序列对于奇异果甜蛋白的合成不是必需的。
