| Summary: | 碩士 === 大葉大學 === 分子生物科技學系碩士班 === 99 === Anti-stress and help sleep peptide Tyr-Leu-Gly-Tyr-Leu-Glu-Gln-Leu-Leu-Arg( YLGYLEQLLR; YLG ) hydrolyzed from casein in milk had proved by the animal experiments and human trials to reduce the production of stress hormones and improve sleep quality ( de Saint Hilaire et al., 2009 )3. In order to establish YLGYLEQLLR peptide production of high-yield and high quantaty, the experiment was performed by using human tyrosine hydroxylase ( HTH ) carrying the 9 copies of functional peptides in the 5 surface domains of this enzyme without affecting its activity ( Goodwill et al., 1998 )5. Each functional peptide with the N-terminal of Lys or Arg and C-terminal of its Arg was designed to facilitate future use of trypsin cutting in the digestion tract to release peptides. The gene completion of the replacement in five surface domains containing anti-stress and help sleep peptide was cloned into the yeast expression vector pYLSC1 with the extracellular secretion and then transformed and integrated into Yarrowia lipolytica chromosome, so we must use the codon usage of Y. lipolytica to yield the highest peptide production finally. In this study, nine copies of peptide were designed in each protein carriers. Each surface domains carried two copies of peptide, except domains 3 and 4 contained 3 copies. Therefore, HTH totally carried 9 copies of functional peptides in the 5 surface domains without affecting its enzymatic activity. After completion of the replacement of each domains, the neighboring domains were connected. The completion of construct is followed by cloning into the Y. lipolytica yeast expression vector pYLSC1 with extracellular secretion, and then finally integrated into Y. lipolytica chromosome. The optimal growth of the yeast strain Y. lipolytica carring 9 copies of functional peptide was characterized as 16 hours in the growth curve with a spectrophotometer. The optimal secretion time of the peptide-carried protein was measured to be at 36 hour stimulation in the induction medium run out glucose content by HPLC. The peptide-carried protein was purified using Ni-NTA column to get rich in anti-stress and help sleep. Subsequent molecular analysis shows that the purified protein is about 65 kDa measured by SDS-PAGE analysis, because the post-translational modification in Y. lipolytica led to the glycosylation of target protein, and the molecular weight of target protein increased in 10 kDa ( Madzak et al., 2004 )9. The quantity of purified protein is 1,025 μg from one litter of yeast culture. The unit activity and the specific activity of purified protein was measured as 7.916×10-2 μmole / min and 3.861 U / mg, respectively. The amino acid sequence of YLGYLEQLLR from the purified protein hydrolysed by trypsin will be analyzed by LC-MS-MS. Finally, the functional analysis of peptide-carried protein was performed in animal experiment and human trials. We hope that the peptide reduce stress and improve the quality of sleep, and then will be active through Government’s healthy food certification mark.
Keywords : anti-stress and help sleep peptide, human tyrosine hydroxylase, Yarrowia lipolytica, specific activity, protein carrier
|
|---|
