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Biological Chemistry

Editor-in-Chief: Brüne, Bernhard

Editorial Board: Buchner, Johannes / Lei, Ming / Ludwig, Stephan / Sies, Helmut / Thomas, Douglas D. / Turk, Boris / Wittinghofer, Alfred

12 Issues per year


IMPACT FACTOR 2017: 3.022

CiteScore 2017: 2.81

SCImago Journal Rank (SJR) 2017: 1.562
Source Normalized Impact per Paper (SNIP) 2017: 0.705

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1437-4315
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Volume 394, Issue 5

Issues

Expression in non-melanogenic systems and purification of soluble variants of human tyrosinase

Petra Cordes
  • Institute of Physiological Chemistry, Philipps University, Karl-von-Frisch-Strasse 1, D-35043 Marburg, Germany
  • Other articles by this author:
  • De Gruyter OnlineGoogle Scholar
/ Wei Sun
  • Institute of Physiological Chemistry, Philipps University, Karl-von-Frisch-Strasse 1, D-35043 Marburg, Germany
  • Institute of Pharmaceutical Chemistry, Philipps University, Marbacher Weg 6, D-35032 Marburg, Germany
  • Other articles by this author:
  • De Gruyter OnlineGoogle Scholar
/ Rainer Wolber / Ludger Kolbe / Gerhard Klebe
  • Institute of Pharmaceutical Chemistry, Philipps University, Marbacher Weg 6, D-35032 Marburg, Germany
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  • De Gruyter OnlineGoogle Scholar
/ Klaus-Heinrich Röhm
  • Corresponding author
  • Institute of Physiological Chemistry, Philipps University, Karl-von-Frisch-Strasse 1, D-35043 Marburg, Germany
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  • Other articles by this author:
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Published Online: 2012-12-13 | DOI: https://doi.org/10.1515/hsz-2012-0300

Abstract

Mammalian tyrosinases are key enzymes of melanin formation. Their native forms undergo complex maturation and sorting processes before being integrated into the melanosomal membrane, which greatly complicates their heterologous expression in other cell types. In the present work, we constructed several differently truncated, soluble variants of human tyrosinase and studied their properties after expression in HEK 293 cells. In addition, we prepared two affinity-tagged forms of the enzyme for expression in the yeast Kluyveromyces lactis and HEK cells, respectively. A Strep-tagged variant was secreted by K. lactis in excellent yields but found to be inactive, whereas a His-tagged variant secreted by HEK 293 cells in an active state could be purified from cell supernatants to near homogeneity. The resulting preparation consisted of an inactive, probably unglycosylated species of about 57 kDa and several glycosylated forms with masses between 63 and 75 kDa, as confirmed by activity staining, Western blotting and mass spectrometry.

Keywords: characterization; expression; human; purification; tyrosinase

About the article

Corresponding author: Klaus-Heinrich Röhm, Institute of Physiological Chemistry, Philipps University, Karl-von-Frisch-Strasse 1, D-35043 Marburg, Germany


Received: 2012-10-12

Accepted: 2012-12-04

Published Online: 2012-12-13

Published in Print: 2013-05-01


Citation Information: Biological Chemistry, Volume 394, Issue 5, Pages 685–693, ISSN (Online) 1437-4315, ISSN (Print) 1431-6730, DOI: https://doi.org/10.1515/hsz-2012-0300.

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