Recombinant strain for tyrosinase production and purification method using the same

Recombinant strain for high-purity human tyrosinase production based on Pichia yeast secretory expression

This technology involves a recombinant Pichia pastoris strain that combines the human tyrosinase or tyrosinase-related protein 1 gene with an AOX1 promoter, alpha-mating factor signal sequence, Twin-Strep-tag, and TEV protease sequence, along with a method for high-purity purification using anion-exchange chromatography.

Most commercially available tyrosinases are mushroom-derived, which have low sequence homology with human enzymes and differ in catalytic activity and substrate specificity, limiting the accuracy of skin-whitening efficacy evaluations. E. coli expression systems cannot perform post-translational modifications, while insect larva systems are time-consuming and costly.

This technology uses a yeast secretory expression system to produce human-type tyrosinase and purifies it through anion-exchange columns and dialysis, securing high-purity enzymes at a lower cost and in less time. It can be applied to screening whitening cosmetic ingredients, researching pigmentary disorders like albinism, and enzyme structural analysis, enabling reliable efficacy evaluations based on human enzymes rather than mushroom enzymes.

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Key Features:

• Recombinant Pichia pastoris SMD1168 strain containing the human tyrosinase gene of SEQ ID NO: 1, an AOX1 promoter, and an alpha-mating factor signal sequence
• Strain producing high-purity tyrosinase with an N-terminus directed toward the protein interior by including both a Twin-Strep-tag sequence and a TEV protease sequence
• Recombinant strain producing high-purity tyrosinase-related protein 1 by combining the human tyrosinase-related protein 1 gene with the same expression elements
• Purification method involving developing the cell culture or lysate of the recombinant strain through anion-exchange column chromatography to obtain an eluate, followed by dialysis

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This invention was developed with support from the Ministry of Education for research on the interaction between human GPCR sweet taste receptors and sweet substances, and for the development of new sweet materials.

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