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    
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.
Thanasis Dalakouras; Brian J Smith; Dimitris Platis; Manon M J Cox; Nikolaos E Labrou
Related Documents :
1719955 - Up-regulation of sodium pump activity in xenopus laevis oocytes by expression of hetero...
12657675 - Phospholemman, a single-span membrane protein, is an accessory protein of na,k-atpase i...
19048685 - Steady-state kinetic analysis of the na+/k+-atpase. the inhibition by potassium and mag...
16954215 - Crystal structure of thrombin in a self-inhibited conformation.
23064115 - Emerging views about the molecular structure of the spliceosomal catalytic center.
22354575 - Fluorescein analogues inhibit seca atpase: the first sub-micromolar inhibitor of bacter...
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    
Laboratory of Enzyme Technology, Department of Agricultural Biotechnology, Agricultural University of Athens, 75 Iera Odos, GR 118 55 Athens, Greece.
Export Citation:
APA/MLA Format     Download EndNote     Download BibTex
MeSH Terms
Affinity Labels
Base Sequence
Cloning, Molecular
DNA Primers
Influenza A Virus, H1N1 Subtype / enzymology,  immunology*
Influenza Vaccines / chemical synthesis*,  immunology
Models, Molecular
Molecular Mimicry
Neuraminidase / genetics,  immunology,  isolation & purification*
Vaccines, Synthetic / immunology*
Reg. No./Substance:
0/Affinity Labels; 0/DNA Primers; 0/Influenza Vaccines; 0/Vaccines, Synthetic; EC

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

Previous Document:  Chiral separation of amino-alcohols using extractant impregnated resins.
Next Document:  Characteristic of theophylline imprinted monolithic column and its application for determination of ...