Cloning, expression and characterization of recombinant sweet‐protein thaumatin II using the methylotrophic yeast Pichia pastoris

Abstract

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.

High-yield Secretion of the Recombinant Sweet-Tasting Protein Thaumatin I

“…In this study, we also determined the threshold value of sweetness for both PreProTH and α-ProTH: 52 ± 17 nM and 60 ± 21 nM, respectively. These results confirm that N-and C-terminal regions of thaumatin play no significant role in the elicitation of sweetness (Masuda et al, 2004) and the attachment of the pro-sequence at the C-terminus does not affect the sweetness of thaumatin.…”

Efficient secretory expression of the sweet-tasting protein brazzein in the yeast Kluyveromyces lactis

“…Recombinant proteins were induced by adding 1% methanol twice a day for 6 days, and approximately 30-90 mg/L was obtained. P. pastoris expression systems have also been used to express the sweet-tasting proteins thaumatin and lysozyme [14][15][16]]. An expression system was also developed using K. lactis, an excellent and well-accepted host for heterologous protein production.…”

Recombinant expressions of sweet plant protein mabinlin II in Escherichia coli and food-grade Lactococcus lactis

“…The sweet protein thaumatin II has been expressed in E. coli and the recombinant protein has been renatured from inclusion bodies to yield a purified protein preparation that is indistinguishable from native thaumatin in all biochemical, spectroscopic, and organoleptic respects (Daniell et al 2000). In addition, thaumatin II was secreted by the methylotrophic yeast Pichia pastoris, and recombinant thaumatin II elicited a sweet taste similar to that of thaumatin II extracted from the Thaumatococcus danielli Benth plants (Masuda et al 2004). The sweet protein monellin was expressed in E. coli, food yeast Candida utilis, and Bacillus subtilis, and a maximum secreted monellin yield of 0.29 mg/mL was extracted from the supernatant of B. subtilis (Kondo et al 1997;Chen et al 2005Chen et al , 2007.…”