Development of High Efficient Systems for Producing Copper Peptide As a Cosmetic Material in the Yeast

Copper peptide is composed of three amino acids (Gly-His-Lys) and copper and naturally in human plasma. Since it was discovered in 1973, it has been reported that copper peptide plays wide roles in activation of wound healing, attraction of immune cells, antioxidant and anti-inflammatory effects, and stimulation of collagen synthesis. Especially, copper peptide is widely used in anti-aging cosmetics. However, copper peptide has been produced by using chemical synthesis. In this study, we developed a biological system to produce and purify this three peptide (Gly-His-Lys) in a GRAS yeast, Pichia pastoris. First, we constructed an expression vector (pBJY-GHK6) in which six of Gly-His-Lys fused to His-tag could be expressed and secreted into media broth. The linearized pBJY-GHK6 was transformed and integrated into genome of P. pastoris and the recombinant strain was cultivated in minimal media (YNB/YSD without adenine). We used an affinity column (Ni-column) to purify the expressed cassettes, [(His)₆-K-(GHK)₆] and [(GHK)₆(His)₆] and treated the expressed peptide with trypsin to generate GHK monomers. We confirmed the expressed peptide on SDS-PAGE and analyzed the GHK monomers by using HPLC and ESI-MS. We are developing optimized condition to increase concentration of the expressed GHK by optimizing culture media, fermentation mode and purification process. In addition, trypsin is going to also be produced in Pichia pastoris and then used in the cleavage of [(His)₆-K-(GHK)₆] and [(GHK)₆(His)₆]. This system will be applied to widely be used in production of cosmetic, cosmeceutical and even pharmaceutical peptides.