title: Efficient production and characterization of the sweet-tasting brazzein secreted by the yeast Pichia pastoris.
abstract: 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.
authors.affiliation: INRA UMR1324, CNRS UMR6265,  Université de Bourgogne , Centre des Sciences du Goût et de l'Alimentation, F-21000 Dijon, France.
authors.forename: Nicolas
authors.initials: N
authors.lastname: Poirier
authors.forename: Natacha
authors.initials: N
authors.lastname: Roudnitzky
authors.forename: Anne
authors.initials: A
authors.lastname: Brockhoff
authors.forename: Christine
authors.initials: C
authors.lastname: Belloir
authors.forename: Marie
authors.initials: M
authors.lastname: Maison
authors.forename: Thierry
authors.initials: T
authors.lastname: Thomas-Danguin
authors.forename: Wolfgang
authors.initials: W
authors.lastname: Meyerhof
authors.forename: Loïc
authors.initials: L
authors.lastname: Briand
mesh_terms: D000595:Amino Acid Sequence; D008969:Molecular Sequence Data; D010843:Pichia / Q000235:genetics / Q000378:metabolism*; D010940:Plant Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D017510:Protein Folding; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D013549:Sweetening Agents / Q000737:chemistry* / Q000378:metabolism*
chemical_list: D010940:Plant Proteins; D011994:Recombinant Proteins; D013549:Sweetening Agents