Avian influenza A/H5N1 neuraminidase expressed in yeast with a functional head domain.
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 72kDa secreted, soluble protein. Glycopeptidase F treatment generated a 45kDa 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.
Yongkiettrakul
S
S
Protein-Ligand Engineering and Molecular Biology Laboratory, BIOTEC Central Research Unit, National Center for Genetic Engineering and Biotechnology, National Science and Technology Development Agency, Pathumthani 12120, Thailand. suganya.yon@biotec.or.th
Boonyapakron
K
K
Jongkaewwattana
A
A
Wanitchang
A
A
Leartsakulpanich
U
U
Chitnumsub
P
P
Eurwilaichitr
L
L
Yuthavong
Y
Y
D000595:Amino Acid Sequence; D015870:Gene Expression; D005814:Genes, Viral; D005822:Genetic Vectors; D006023:Glycoproteins / Q000302:isolation & purification / Q000378:metabolism; D053124:Influenza A Virus, H5N1 Subtype / Q000201:enzymology*; D007700:Kinetics; D008969:Molecular Sequence Data; D009439:Neuraminidase / Q000302:isolation & purification / Q000378:metabolism*; D010843:Pichia / Q000378:metabolism; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism*; D014170:Transformation, Genetic
D006023:Glycoproteins; D011994:Recombinant Proteins; D009439:Neuraminidase