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- Title
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.
- Authors
Cai, Chenggu; Li, Lei; Lu, Nan; Zheng, Weiwei; Yang, Liu; Liu, Bo
- 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.
- Subjects
MONELLIN; HEAT stability in proteins; PICHIA pastoris; SWEETENERS; MUTAGENESIS; CHROMATOGRAPHIC analysis; ESCHERICHIA coli
- Publication
Biotechnology Letters, 2016, Vol 38, Issue 11, p1941
- ISSN
0141-5492
- Publication type
Article
- DOI
10.1007/s10529-016-2182-4