Efficient Production and Characterization of the Sweet-Tasting Brazzein Secreted by the Yeast Pichia pastoris
详细信息 查看全文
- 作者：Nicolas Poirier ; Natacha Roudnitzky ; Anne Brockhoff ; Christine Belloir ; Marie Maison ; Thierry Thomas-Danguin ; Wolfgang Meyerhof ; Lo茂c Briand
- 刊名：Journal of Agricultural and Food Chemistry
- 出版年：2012
- 出版时间：October 3, 2012
- 年：2012
- 卷：60
- 期：39
- 页码：9807-9814
- 全文大小：343K
- 年卷期：v.60,no.39(October 3, 2012)
- ISSN：1520-5118
文摘
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 1H 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.