Purification of Recombinant Human Tyrosinase from Insect Larvae Infected with the Baculovirus Vector

Monika B. Dolinska1, Paul T. Wingfield2, Yuri V. Sergeev1

1 National Eye Institute, National Institutes of Health, Bethesda, Maryland, 2 National Institute of Arthritis and Musculoskeletal and Skin Diseases, National Institutes of Health, Bethesda, Maryland
Publication Name:  Current Protocols in Protein Science
Unit Number:  Unit 6.15
DOI:  10.1002/cpps.37
Online Posting Date:  August, 2017
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The purification of an enzyme from insect larvae infected with a baculovirus vector is described. The enzyme tyrosinase is of biomedical importance and catalyzes the first rate‐limiting steps in melanin production. Tyrosinase mutations can result in oculocutaneous albinism type 1 (OCA1), an inherited eye disease associated with decreased melanin pigment production and vision defects. To simplify expression and subsequent purification, the extracellular domain is expressed in insect cells, produced in Trichoplusia ni larvae, and purified using affinity and size‐exclusion chromatography. The purified recombinant human tyrosinase is a soluble monomeric glycoprotein with an activity that mirrors the tyrosinase in vivo function. © 2017 by John Wiley & Sons, Inc.

Keywords: human tyrosinase; oculocutaneous albinism type 1; larval biomass; affinity chromatography; size‐exclusion chromatography; enzymatic activity

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Table of Contents

  • Introduction
  • Basic Protocol 1: Intra‐Melanosomal Tyrosinase Domain Expression and Purification
  • Support Protocol 1: Measurement of Tyrosinase Enzymatic Activity
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
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Basic Protocol 1: Intra‐Melanosomal Tyrosinase Domain Expression and Purification

  • T. ni larvae biomass containing the protein of interest (e.g., Allotropic Tech)
  • Lysis buffer (see recipe)
  • Binding buffer (see recipe)
  • Elution buffer (see recipe)
  • Imidazole
  • Gel‐filtration buffer (see recipe)
  • Anti‐tyrosinase antibody (e.g., Santa Cruz Biotechnology T311)
  • Anti‐His antibody (e.g., Thermo Fisher Scientific)
  • 50‐ml conical tubes
  • Tissue homogenizer (e.g., Omni Tissue Homogenizer TH) and probes (e.g., Hard Tissue Omni Tip Homogenizer Probes)
  • Laboratory rotator (e.g., Boekel Scientific Orbitron Rotator)
  • Ultrasonic processor
  • 50‐ml high‐speed, round‐bottom centrifuge tubes (e.g., Thermo Scientific)
  • Centrifuge (e.g., Sorvall Lynx 4000) with fixed‐angle rotor (e.g., Fiberlite F21‐8 × 50y)
  • UV‐Vis spectrophotometer (e.g., NanoDrop 2000c)
  • Immobilized metal affinity chromatography (IMAC) column (e.g., GE Healthcare HisTrap FF Crude)
  • Chromatography system (e.g., Bio‐Rad BioLogic DuoFlow System)
  • Dialysis membranes, 10K molecular weight cutoff (e.g., SnakeSkin Dialysis Tubing or Slide‐A‐Lyzer Dialysis Cassettes)
  • Magnetic stir plate and stir bars
  • Gel‐filtration columns:
  • HiPrep 16/60 Sephacryl S‐100 (e.g., GE Healthcare)
  • Superdex 75 10/300 (e.g., GE Healthcare)
  • Gel analysis software (e.g., Silk Scientific, UN‐SCAN‐IT gel)
  • Additional materials and equipment for SDS‐PAGE (unit 10.1; Gallagher, ), Western blot (unit 10.8; Goldman, Harper, & Speicher, ), MALDI‐TOF/TOF (unit 16.1; Jiménez, Huang, Qui, & Burlingame, ), and sedimentation assays (unit 20.1; Sergeev, Dolinska, & Wingfield, )

Support Protocol 1: Measurement of Tyrosinase Enzymatic Activity

  • 3,4‐dihydroxy‐L‐phenylalanine (L‐DOPA; e.g., Sigma‐Aldrich)
  • 10 mM sodium phosphate buffer (see appendix 2E),
  • Sample containing tyrosinase (see protocol 1Basic Protocol)
  • 96‐well polystyrene plates, clear‐bottomed
  • 37°C incubator
  • Microplate reader (e.g., SpectraMax i3 Multi‐Mode Microplate Detection Platform)
  • Additional reagents and equipment for enzymatic assays and determining Michaelis‐Menton kinetics (see unit 3.5; Allison, )
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Literature Cited

Literature Cited
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