Cloning, expression and characterization of recombinant sweet-protein thaumatin II using the methylotrophic yeast Pichia pastoris.
Thaumatin, an intensely sweet-tasting protein, was secreted by the methylotrophic yeast Pichia pastoris. The mature thaumatin II gene was directly cloned from Taq polymerase-amplified PCR products by using TA cloning methods and fused the pPIC9K expression vector that contains Saccharomyces cerevisiae prepro alpha-mating factor secretion signal. Several additional amino acid residues were introduced at both the N- and C-terminal ends by genetic modification to investigate the role of the terminal end region for elicitation of sweetness in the thaumatin molecule. The secondary and tertiary structures of purified recombinant thaumatin were almost identical to those of the plant thaumatin molecule. Recombinant thaumatin II elicited a sweet taste as native plant thaumatin II; its threshold value of sweetness to humans was around 50 nM, which is the same as that of plant thaumatin II. These results demonstrate that the functional expression of thaumatin II was attained by Pichia pastoris systems and that the N- and C-terminal regions of the thaumatin II molecule do not -play an important role in eliciting the sweet taste of thaumatin.
Division of Food Science and Biotechnology, Graduate School of Agriculture, Kyoto University, Uji, Kyoto 611-0011, Japan.
Tetsuya
T
Masuda
Shinobu
S
Tamaki
Ryosuke
R
Kaneko
Ritsuko
R
Wada
Yuki
Y
Fujita
Alka
A
Mehta
Naofumi
N
Kitabatake
D019943:Amino Acid Substitution; D003001:Cloning, Molecular / Q000379:methods; D015966:Gene Expression Regulation, Fungal / Q000502:physiology; D006801:Humans; D010843:Pichia / Q000235:genetics* / Q000378:metabolism*; D010940:Plant Proteins / Q000032:analysis* / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D015202:Protein Engineering / Q000379:methods; D011994:Recombinant Proteins / Q000032:analysis / Q000096:biosynthesis / Q000737:chemistry / Q000145:classification; D013549:Sweetening Agents / Q000032:analysis* / Q000737:chemistry / Q000145:classification / Q000378:metabolism*; D013649:Taste
D010940:Plant Proteins; D011994:Recombinant Proteins; D013549:Sweetening Agents; C057542:thaumatin II protein, Thaumatococcus daniellii