Immunoaffinity Purification of the Class V Chitin Synthase of Wangiella (Exophiala) dermatitidis

Abstract

The class V chitin synthase is unique because it has a myosin motor-like domain fused to its catalytic domain. The biochemical properties of this enzyme and its function remain undefined beyond the knowledge that it is the only single chitin synthase required for sustained cell growth at elevated temperatures and, consequently, virulence. This report describes our successful efforts to isolate and purify an active and soluble form of the enzyme from the cell membranes of Wangiella by using a specific polyclonal antibody. To our knowledge, this is the first purification of a single chitin synthase of a filamentous fungus.

Keywords:

• Black pathogenic fungus
• Chitin synthase
• Immunoaffinity purification
• Transmembrane protein

ACKNOWLEDGMENTS

We thank Tuan Vo for the valuable technical assistance he provided during the course of this work. This research was supported by a grant to P. J. S. from the National Institute of Allergy and Infectious Diseases (AI33049).

Notes

∗Units are defined as µmol of ¹⁴C-labeled UDP-GlcNAc incorporated in 1 min into insoluble chitin product.

Data represent the means of at least three independent reactions. Standard error of CPM results ±5%. The CPM results represent the activity minus background from assays without protein in reaction mixtures. In all steps, the chitin synthase activity was measured after activation with optimal amounts of trypsin.