The cleavage of the hemagglutinin protein of H5N2 avian influenza virus in yeast

Wang, Chi Y.; Luo, Yu; Chen, Yu T.; Li, Shu K.; Lin, Chi; Hsieh, Yao Ching; Liu, Hung-Jen

doi:10.1016/j.jviromet.2007.07.012

Cited by 15 publications

(9 citation statements)

References 40 publications

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“…The AFM image of the recombinant HA protein shown in Figs. 4 and 5 compares well with previous structural studies on HA protein Klenk et al 1975;Lazarowitz and Choppin 1975;Wang et al 2007). The AFM scanning images of the purified recombinant HA protein showed that the two processed HA 1 and HA 2 subunits are linked (Figs.…”

Section: Discussionsupporting

confidence: 87%

“…Clone 1 was the negative control S. pombe containing the pNMT1 empty vector. HAU represent the unit of hemagglutining activity HA 2 subunit has hydrophobic N-terminal fusion peptide anchored into lipid bilayer by its C-terminal transmembrane domain Klenk et al 1975;Lazarowitz and Choppin 1975;Wang et al 2007). The AFM image of the recombinant HA protein shown in Figs.…”

Section: Discussionmentioning

confidence: 99%

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Integrity of a recombinant hemagglutinin protein of an avian influenza virus

Williams

Singh

et al. 2009

Biotechnol Lett

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An open reading frame representing cDNA from a hemagglutinin (HA) encoding gene of a low pathogenic avian influenza virus (AIV) subtype H10N7 was cloned in the pNMT1-TOPO vector under the control of thiamine response promoter. This construct was designated as pNMT1-HA. The pNMT1-HA construct was transformed into Schizosaccharomyces pombe for expression of HA antigen. The correct expression of recombinant HA protein was confirmed by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot. The level of expression of recombinant HA protein was approximately 0.2% of total soluble protein. Purified yeast-derived recombinant HA protein showed hemagglutination activity. The 2-D and 3-D scanning images of recombinant HA protein were observed with an atomic force microscope (AFM). The structural integrity of the HA protein under AFM and hemagglutination activity provided support that the recombinant HA protein may be suitable for development of AIV subunit vaccine for mass administration to poultry.

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Section: Discussionsupporting

confidence: 87%

Section: Discussionmentioning

confidence: 99%

Integrity of a recombinant hemagglutinin protein of an avian influenza virus

Williams

Singh

et al. 2009

Biotechnol Lett

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“…In addition, P. pastoris is potentially adaptable for safe and inexpensive large-scale fermentation. P. pastoris expression system has been used to produce a number of viral proteins, including hepatitis virus proteins (Chen, 2007;Han et al, 2006), HIV-1 envelope glycoprotein (Scorer et al, 1993), viral capsid antigen of Epstein-Barr virus (Hu et al, 2007), influenza virus RNA polymerase (Hwang et al, 2000), SARS coronavirus nucleocapsid protein (Liu et al, 2005), dengue virus envelope protein (Valdés et al, 2007), rubella virus glycoprotein E1 (Wen and Wang, 2005), and hemagglutinins of avian influenza virus H5, H7, and H9 subtypes (Wang et al, 2007;Xu et al, 2006).…”

Section: Introductionmentioning

confidence: 99%

Avian influenza A/H5N1 neuraminidase expressed in yeast with a functional head domain

Yongkiettrakul

Boonyapakron

Jongkaewwattana

et al. 2009

Journal of Virological Methods

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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.

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“…Although a few reports have shown that full-length influenza HA0 can be expressed on the yeast surface, detailed information was not provided as to whether the yeast-displayed HA was folded into its correct native conformation [9][10][11]. In this study, the fulllength extracellular HA0 protein and HA1 and HA2 subunits were cloned into a yeast surface expression vector (Fig.…”

Section: Discussionmentioning

confidence: 99%

Fine epitope mapping of monoclonal antibodies against hemagglutinin of a highly pathogenic H5N1 influenza virus using yeast surface display

Han

Sui

Bennett

et al. 2011

Biochemical and Biophysical Research Communications

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Highly pathogenic H5N1 avian influenza viruses pose a debilitating pandemic threat. Thus, understanding mechanisms of antibody-mediated viral inhibition and neutralization escape is critical. Here, a robust yeast display system for fine epitope mapping of viral surface hemagglutinin (HA)-specific antibodies is demonstrated. The full-length H5 subtype HA (HA0) was expressed on the yeast surface in a correctly folded conformation, determined by binding of a panel of extensively characterized neutralizing human monoclonal antibodies (mAbs). These mAbs target conformationally-dependent epitopes of influenza A HA, which are highly conserved across H5 clades and group 1 serotypes. By separately displaying HA1 and HA2 subunits on yeast, domain mapping of two anti-H5 mAbs, NR2728 and H5-2A, localized their epitopes to HA1. These anti-H5 mAb epitopes were further fine mapped by using a library of yeast-displayed HA1 mutants and selecting for loss of binding without prior knowledge of potential contact residues. By overlaying key mutant residues that impacted binding onto a crystal structure of HA, the NR2728 mAb was found to interact with a fully surface-exposed contiguous patch of residues at the receptor binding site (RBS), giving insight into the mechanism underlying its potent inhibition of virus binding. The non-neutralizing H5-2A mAb was similarly mapped to a highly conserved H5 strain-specific but poorly accessible location on a loop at the trimer HA interface. These data further augment our toolchest for studying HA antigenicity, epitope diversity and accessibility in response to natural and experimental influenza infection and vaccines.

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The cleavage of the hemagglutinin protein of H5N2 avian influenza virus in yeast

Cited by 15 publications

References 40 publications

Integrity of a recombinant hemagglutinin protein of an avian influenza virus

Integrity of a recombinant hemagglutinin protein of an avian influenza virus

Avian influenza A/H5N1 neuraminidase expressed in yeast with a functional head domain

Fine epitope mapping of monoclonal antibodies against hemagglutinin of a highly pathogenic H5N1 influenza virus using yeast surface display

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