National Science & Technology Development Agency - Thailand; National Center Genetic Engineering & Biotechnology (BIOTEC); National Science & Technology Development Agency - Thailand; National Center Genetic Engineering & Biotechnology (BIOTEC); National Science & Technology Development Agency - Thailand; National Center Genetic Engineering & Biotechnology (BIOTEC)
Type
Article
Source Title
JOURNAL OF VIROLOGICAL METHODS
ISSN
0166-0934
Year
2009
Volume
156
Issue
11
Open Access
Green Published, Bronze
Publisher
ELSEVIER SCIENCE BV
DOI
10.1016/j.jviromet.2008.10.025
Format
PDF
Abstract
The study reports heterologous expression in Pichia pastoris of active neuraminidase derived from avian influenza virus A/Viet Nam/DT-036/2005(H5N1). A gene encoding the neuraminidase N1 head domain (residues 63-449) was fused directly in-frame with the Saccharomyces cerevisiae alpha-factor secretion signal in pPICZ(A vector. Recombinant N1 neuraminidase was expressed in P pastoris as a 72 kDa secreted, soluble protein. Glycopeptidase F treatment generated a 45 kDa product, indicating that the secreted recombinant N1 neuraminidase is an N-linked glycoprotein. Kinetic studies and inhibition tests with oseltamivir carboxylate demonstrated that the recombinant N1 neuraminidase has similar K-m and K-i values to those of the viral N1 neuraminidase. This yeast-based heterologous expression system provided functionally active recombinant N1 neuraminidase that should be useful in anti-influenza drug screening, and also as a potential protein-based vaccine. (c) 2008 Elsevier B.V. All rights reserved.
