Cloning of the thaumatin I cDNA and characterization of recombinant thaumatin I secreted by Pichia pastoris
Cloning of the thaumatin I cDNA and characterization of recombinant thaumatin I secreted by Pichia pastoris
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DOI：
10.1021/bp070072v
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发表时间：
2007-09-01
影响因子：
2.9
通讯作者：
Kitabatake, Naofumi
中科院分区：
文献类型：
--
作者：
Ide, Nobuyuki;Kaneko, Ryosuke;Kitabatake, Naofumi
奇异果甜蛋白是一种甜味蛋白质，包含一些变体的混合物。主要的变体是thaacetin I和II。尽管索马甜I的氨基酸序列已为人所知，索马甜H的cDNA的核苷酸序列也已被阐明，但索马甜I的核苷酸序列一直存在争议。我们从丹氏奇异果果实中克隆了两个奇异果甜蛋白cDNA。一个是与已报道的thaurnatin H相同的核苷酸序列，另一个是新的核苷酸序列。从新cDNA推导的氨基酸序列与索马甜1的氨基酸序列相同，唯一的例外是113位的残基（Asp而不是Asn），这表明新的索马甜cDNA是索马甜I的cDNA。将该thaurnatin I cDNA转化到毕赤酵母X-33中，表达的重组thaurnatin I进行纯化和表征。重组索马甜蛋白I的甜度阈值与植物索马甜蛋白1的甜度阈值相同，尽管在重组索马甜蛋白I的N-末端连接了几个意想不到的氨基酸残基。这些表明，thaurnatin的N-末端部分对于甜味的引发不是关键的。
Thaumatin is a sweet-tasting protein comprising a mixture of some variants. The major variants are thaumatins I and II. Although the amino acid sequence of thaumatin I was known and the nucleotide sequence of cDNA of thaumatin H was elucidated, the nucleotide sequence of thaurnatin I has been controversial. We have cloned two thaumatin cDNAs from the fruit of Thaumatococcus daniellii Benth. One is the same nucleotide sequence as that of thaurnatin H already reported, and the other is a novel nucleotide sequence. The amino acid sequence deduced from the novel cDNA was the same amino acid sequence as that of thaumatin 1, the only exception being the residue at position 113 (Asp instead of Asn), indicating that the novel thaumatin cDNA is that for thaumatin I. This thaurnatin I cDNA was transformed into Pichia pastoris X-33, and the recombinant thaurnatin I expressed was purified and characterized. The threshold value of sweetness of the recombinant thaurnatin I was the same as that of the plant thaurnatin 1, although several unexpected amino acid residues were attached to the N-terminal of the recombinant thaumatin I. These indicate that the N-terminal portion of thaurnatin, is not critical for the elicitation of sweetness.