Efficient production and characterization of the sweet-tasting brazzein secreted by the yeast Pichia pastoris.
Efficient production and characterization of the sweet-tasting brazzein secreted by the yeast Pichia pastoris.
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DOI：
10.1021/jf301600m
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发表时间：
2012-09
影响因子：
6.1
通讯作者：
Nicolas Poirier;N. Roudnitzky;A. Brockhoff;C. Belloir;Marie Maison;T. Thomas-Danguin;W. Meyerhof;L. Briand
中科院分区：
文献类型：
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作者：
Nicolas Poirier;N. Roudnitzky;A. Brockhoff;C. Belloir;Marie Maison;T. Thomas-Danguin;W. Meyerhof;L. Briand
Brazzein是一种小的、热稳定和pH稳定的甜蛋白，存在于西非植物Pentadiplandra brazzeana Baillon的果实中。它以两种不同的甜味强度存在。主要形式称为pyrE-bra，在其N-末端含有焦谷氨酸，而次要形式称为des-pyrE-bra，缺少该残基。在这里，我们描述了异源表达在甲醇营养型酵母巴斯德毕赤酵母的两种天然形式的植物甜蛋白，pyrE-bra和des-pyrE-bra，和一个额外的形式，称为Q1-bra，这是不是天然存在的水果。Q1-bra与pyrE-bra的不同之处在于在其N-末端具有谷氨酰胺残基而不是pyrE。在6天的表达期内，我们分别获得了约90、30和90 mg/L的纯化的重组pyrE-bra、Q1-bra和des-pyrE-bra植物甜蛋白形式。纯化重组蛋白并进行质谱分析和（1）1H NMR光谱分析。数据表明重组植物甜蛋白形式被正确折叠。此外，它们在体外激活人类甜味受体，并在体内诱发甜味，其性质与两种天然植物甜蛋白形式相似。
Brazzein is a small, heat-, and pH-stable sweet protein present in the fruits of the West African plant Pentadiplandra brazzeana Baillon. It exists in two forms differing in sweetness intensity. The major form, called pyrE-bra, contains a pyroglutamic acid at its N-terminus, while the minor form, called des-pyrE-bra, lacks this residue. Here we describe the heterologous expression in the methylotrophic yeast Pichia pastoris of two natural forms of brazzein, pyrE-bra and des-pyrE-bra, and an additional form, called Q1-bra, which is not naturally occurring in the fruit. Q1-bra differs from pyrE-bra in having a glutamine residue instead of pyrE at its N-terminus. Over an expression period of 6 days, we obtained approximately 90, 30, and 90 mg/L of purified recombinant pyrE-bra, Q1-bra, and des-pyrE-bra brazzein forms, respectively. Recombinant proteins were purified and submitted to mass spectrometry and (1)H NMR spectroscopy. The data indicate that the recombinant brazzein forms were properly folded. Moreover, they activated the human sweet receptor in vitro and evoked sweetness in vivo with properties similar to those of the two natural brazzein forms.