Abstract
Objectives
To improve the stability and sweetness of the sweet-tasting protein, monellin, by using site-directed mutagenesis and a Pichia pastoris expression system with a GAPDH constitutive promoter.
Results
Both wild-type and E2 N mutant of single-chain monellin gene were cloned into the PGAPZαA vector and expressed in Pichia pastoris. The majority of the secreted recombinant protein, at 0.15 g/l supernatant, was monellin. This was purified by Sephadex G50 chromatography. The sweetness threshold of wild-type and E2 N were 30 μg/ml and 20 μg/ml, respectively. Compared with the proteins expressed in Escherichia coli, the thermostability of both proteins was improved. The N-terminal sequence is determinative for the sweetness of the proteins expressed in yeast strains.
Conclusions
Site-directed mutagenesis, modification of the N-terminus of monellin, and without the need of methanol induction in P. pastoris expression system, indicate the possibility for large-scale production of this sweet-tasting protein.
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Acknowledgments
This work was supported by the Natural Science Foundation of China (31271118 and 31501450).
Supporting Information
Supplemental Fig. 1—The three-dimensional structure of monellin (PDB:1IV9). The α-helix, β-sheet and β-loop are colored in red, yellow and green, respectively. The mutated residue Glu 2 at the protein surface is labeled, rendered as sticks and colored by atomic types.
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Cai, C., Li, L., Lu, N. et al. Expression of a high sweetness and heat-resistant mutant of sweet-tasting protein, monellin, in Pichia pastoris with a constitutive GAPDH promoter and modified N-terminus. Biotechnol Lett 38, 1941–1946 (2016). https://doi.org/10.1007/s10529-016-2182-4
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DOI: https://doi.org/10.1007/s10529-016-2182-4