June 2017
Volume 58, Issue 8
Open Access
ARVO Annual Meeting Abstract  |   June 2017
Purification and Characterization of Recombinant Human Tyrosinase Related Protein 1
Author Affiliations & Notes
  • Kenneth Lee Young II
    National Eye Institute, National Institutes of Health, Bethesda, Maryland, United States
  • Monika Dolinksa
    National Eye Institute, National Institutes of Health, Bethesda, Maryland, United States
  • Paul Wingfield
    National Institute of Arthritis and Musculoskeletal and Skin Diseases, National Institutes of Health, Bethesda, Maryland, United States
  • Eugenia Poliakov
    National Eye Institute, National Institutes of Health, Bethesda, Maryland, United States
  • Yuri V Sergeev
    National Eye Institute, National Institutes of Health, Bethesda, Maryland, United States
  • Footnotes
    Commercial Relationships   Kenneth Young II, None; Monika Dolinksa, None; Paul Wingfield, None; Eugenia Poliakov, None; Yuri Sergeev, None
  • Footnotes
    Support  None
Investigative Ophthalmology & Visual Science June 2017, Vol.58, 626. doi:
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      Kenneth Lee Young II, Monika Dolinksa, Paul Wingfield, Eugenia Poliakov, Yuri V Sergeev; Purification and Characterization of Recombinant Human Tyrosinase Related Protein 1. Invest. Ophthalmol. Vis. Sci. 2017;58(8):626.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose : Human Tyrosinase Related Protein 1 (hTyrp1) is involved in the melanogenesis pathway and various mutations in its corresponding gene (TYRP1) have been linked to rufous albinism, also known as oculocutaneous albinism type 3 (OCA3), an autosomal recessive disorder. While hTyrp1 is an abundant glycoprotein expressed within melanocytes, the role/s in humans remains mostly enigmatic. Currently efforts to unravel hTyrp1 function have been restricted to melanocyte and murine model studies. Here, we purified and characterized recombinant hTyrp1 as well as the intra-melanosomal domain of human tyrosinase related protein 1 (hTyrp1tr).

Methods : Whole Trichoplusia ni (T. ni) larvae contained individually expressed recombinant hTyrp1 and hTyrp1tr. The membrane bound hTyrp1 was solubilized in the presence of 1% of Fos-Choline-12 (Fos-C). The IMAC and size exclusion chromatography (SEC) were used in the purification process, where all buffers contained 0.1% Fos-C. hTyrp1tr was purified in the absence of detergent. The SEC and sedimentation equilibrium were used to access the oligomeric state of both proteins. In the presence and absence of 8M Urea, tryptophan fluoresence was measured to determine conformational state.

Results : Protein identity was confirmed using specific antibodies and mass spectrometry. The hTyrp1 polypeptide molecular weight of approximately 65 to 70 kDa was displayed through SDS-PAGE and confirmed by Western-Blot. The range of molecular mass is attributed to the variability in the protein glycosylation. In the SEC profile both, hTyrp1 and hTyrp1tr, were eluted as single peaks of monomeric proteins, at about 105 kDa and 63 kDA, respectively. The monomeric state of hTryp1tr was confirmed using sedimentation equilibrium. Tryptophan fluoresence demonstrates purified hTyrp1tr is in a folded conformational state. Both hTyrp1 and hTryp1tr fail to show oxidase activity yielding indole-2-carboxylic acid-5,6-quinone (IDCA) from dihydroxyindole-2-carboxylic acid (DHICA); although this activity has been confirmed in murine models.

Conclusions : Full-length hTyrp1 and hTyrp1tr were successfully overexpressed in T. ni larvae, solubilized, and purified. Purified recombinant hTryp1 will further elucidate its function in human melanocytes. Moreover, it remains pivotal for the search of suitable activators of mutant variants in treatment of genetic disorders, such as oculocutaneous albinism type 3.

This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.

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