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.
abstract: OBJECTIVE
  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.
authors: Department of Bioengineering, Qilu University of Technology, Jinan, 250353, Shandong, People's Republic of China.
authors: Chenggu
authors: C
authors: Cai
authors: Department of Bioengineering, Qilu University of Technology, Jinan, 250353, Shandong, People's Republic of China.
authors: Lei
authors: L
authors: Li
authors: Department of Bioengineering, Qilu University of Technology, Jinan, 250353, Shandong, People's Republic of China.
authors: Nan
authors: N
authors: Lu
authors: Department of Food Science and Engineering, Qilu University of Technology, Jinan, 250353, Shandong, People's Republic of China.
authors: Weiwei
authors: W
authors: Zheng
authors: Department of Food Science and Engineering, Qilu University of Technology, Jinan, 250353, Shandong, People's Republic of China.
authors: Liu
authors: L
authors: Yang
authors: Department of Food Science and Engineering, Qilu University of Technology, Jinan, 250353, Shandong, People's Republic of China. ertrdfgg@qlu.edu.cn.
authors: Bo
authors: B
authors: Liu
mesh_terms: D000595:Amino Acid Sequence; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000235:genetics*; D006358:Hot Temperature; D016297:Mutagenesis, Site-Directed; D010843:Pichia / Q000235:genetics*; D010940:Plant Proteins / Q000235:genetics / Q000378:metabolism*; D011401:Promoter Regions, Genetic; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism
chemical_list: D010940:Plant Proteins; D011994:Recombinant Proteins; C002541:monellin protein, Dioscoreophyllum cumminsii; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases
keywords: Monellin; Pichia pastoris; Site-directed mutagenesis; Sweet-tasting protein; Sweetness threshold; Thermostability