title: Efficient secretory expression of the sweet-tasting protein brazzein in the yeast Kluyveromyces lactis.
abstract: Brazzein is an intensely sweet-tasting protein with high water solubility, heat stability, and taste properties resembling those of carbohydrate sweeteners. In the present study, we describe the expression of the synthetic gene encoding brazzein, a sweet protein in the yeast Kluyveromyces lactis. The synthetic brazzein gene was designed based on the biased codons of the yeast, so as to optimize its expression, as well as on the extracellular secretion for expression in an active, soluble form. The synthesized brazzein gene was cloned into the secretion vector pKLAC2, which contains the yeast prepropeptide signal from the Saccharomycescerevisiae α-mating factor. The constructed plasmid pKLAC2-des-pE1M-brazzein was introduced into the yeast K. lactis GG799. The yeast transformants were cultured for high-yield secretion of the recombinant des-pE1M-brazzein in YPGal medium for 96 h at 30°C. The expressed recombinant des-pE1M-brazzein was purified by CM-Sepharose column chromatography and approximately 104 mg/L was obtained. The purity and conformational state of the recombinant des-pE1M-brazzein were confirmed using SDS-PAGE, HPLC, and circular dichroism. The identity of the recombinant protein was also confirmed by N-terminal amino acid analysis and taste testing. The purified recombinant des-pE1M-brazzein had an intrinsic sweetness in its minor form, approximately 2130 times sweeter than sucrose on a weight basis. These results demonstrate that the K. lactis expression system is useful for producing the recombinant brazzein in active form at a high yield with attributes useful in the food industry.
authors.affiliation: Laboratory of Biomolecular Chemistry, Department of Chemistry, College of Natural Sciences, Chung-Ang University, Dongjak-Ku, Seoul, Republic of Korea.
authors.forename: Hyun-Joo
authors.initials: HJ
authors.lastname: Jo
authors.forename: Jin-Seok
authors.initials: JS
authors.lastname: Noh
authors.forename: Kwang-Hoon
authors.initials: KH
authors.lastname: Kong
mesh_terms: D003062:Codon; D004591:Electrophoresis, Polyacrylamide Gel; D007716:Kluyveromyces / Q000378:metabolism*; D010940:Plant Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism; D013549:Sweetening Agents / Q000737:chemistry
chemical_list: D003062:Codon; D010940:Plant Proteins; D011994:Recombinant Proteins; D013549:Sweetening Agents
keywords: Brazzein; Kluyveromyces lactis; Recombination; Secretory expression; Sweet protein; Yeast