Document Detail


Development of recombinant protein-based influenza vaccine. Expression and affinity purification of H1N1 influenza virus neuraminidase.
MedLine Citation:
PMID:  17046775     Owner:  NLM     Status:  MEDLINE    
Abstract/OtherAbstract:
The influenza virus surface glycoprotein antigen neuraminidase (NA) is a crucial viral enzyme with many potential medical applications; therefore, the development of efficient upstream and downstream processing strategy for the expression and purification of NA is of high importance. In the present work the NA gene from the H1N1 influenza virus strain A/Beijing/262/95 was cloned from viral RNA and expressed in expresSF+ insect cells using the baculovirus expression vector system (BVES). A limited affinity-ligand library was synthesized and evaluated for its ability to bind and purify the recombinant H1N1 neuraminidase. Affinity-ligand design was based on mimicking the interactions of the lock-and-key (LAK) motif (Phe-Gly-Gln), a common structural moiety found in the subunit interface of glutathione S-transferase I (GST I), and plays an important structural role in subunit-subunit recognition. Solid-phase combinatorial chemistry was used to synthesize 13 variants of the lock-and-key lead ligand (Phe-Trz-X, where X was selected alpha-amino acid) using the 1,3,5-triazine moiety (Trz) as the scaffold for assembly. One immobilized ligand, bearing phenylalanine and isoleucine linked on the chlorotriazine ring (Phe-Trz-Ile), displayed high affinity for NA. Absorption equilibrium and molecular modeling studies were carried out to provide a detailed picture of Phe-Trz-Ile interaction with NA. This LAK-mimetic affinity adsorbent was exploited in the development of a facile purification protocol for NA, which led to 335-fold purification in a single-step. The present purification procedure is the most efficient reported so far for recombinant NA.
Authors:
Thanasis Dalakouras; Brian J Smith; Dimitris Platis; Manon M J Cox; Nikolaos E Labrou
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Publication Detail:
Type:  Journal Article; Research Support, Non-U.S. Gov't     Date:  2006-10-16
Journal Detail:
Title:  Journal of chromatography. A     Volume:  1136     ISSN:  0021-9673     ISO Abbreviation:  J Chromatogr A     Publication Date:  2006 Dec 
Date Detail:
Created Date:  2006-11-20     Completed Date:  2007-01-24     Revised Date:  2009-01-15    
Medline Journal Info:
Nlm Unique ID:  9318488     Medline TA:  J Chromatogr A     Country:  Netherlands    
Other Details:
Languages:  eng     Pagination:  48-56     Citation Subset:  IM    
Affiliation:
Laboratory of Enzyme Technology, Department of Agricultural Biotechnology, Agricultural University of Athens, 75 Iera Odos, GR 118 55 Athens, Greece.
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MeSH Terms
Descriptor/Qualifier:
Adsorption
Affinity Labels
Animals
Base Sequence
Cloning, Molecular
DNA Primers
Influenza A Virus, H1N1 Subtype / enzymology,  immunology*
Influenza Vaccines / chemical synthesis*,  immunology
Insects
Models, Molecular
Molecular Mimicry
Neuraminidase / genetics,  immunology,  isolation & purification*
Vaccines, Synthetic / immunology*
Chemical
Reg. No./Substance:
0/Affinity Labels; 0/DNA Primers; 0/Influenza Vaccines; 0/Vaccines, Synthetic; EC 3.2.1.18/Neuraminidase

From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine


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