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@ARTICLE{Hung:151099,
      author       = {Hung, Yu-Fu and Valdau, Olga and Schünke, Sven and Stern,
                      O and Koenig, Bernd W. and Willbold, Dieter and Hoffmann,
                      Silke},
      title        = {{R}ecombinant {P}roduction of the {A}mino {T}erminal
                      {C}ytoplasmic {R}egion of {D}engue {V}irus
                      {N}on-{S}tructural {P}rotein 4{A} for {S}tructural
                      {S}tudies},
      journal      = {PLoS one},
      volume       = {9},
      number       = {1},
      issn         = {1932-6203},
      address      = {Lawrence, Kan.},
      publisher    = {PLoS},
      reportid     = {FZJ-2014-01121},
      pages        = {e86482 (1 - 10)},
      year         = {2014},
      abstract     = {BACKGROUND:Dengue virus (DENV) is a mosquito-transmitted
                      positive single strand RNA virus belonging to the
                      Flaviviridae family. DENV causes dengue fever, currently the
                      world's fastest-spreading tropical disease. Severe forms of
                      the disease like dengue hemorrhagic fever and dengue shock
                      syndrome are life-threatening. There is no specific
                      treatment and no anti-DENV vaccines. Our recent data
                      suggests that the amino terminal cytoplasmic region of the
                      dengue virus non-structural protein 4A (NS4A) comprising
                      amino acid residues 1 to 48 forms an amphipathic helix in
                      the presence of membranes. Its amphipathic character was
                      shown to be essential for viral replication. NMR-based
                      structure-function analysis of the NS4A amino terminal
                      region depends on its milligram-scale production and
                      labeling with NMR active isotopes.METHODOLOGY/PRINCIPAL
                      FINDINGS:This report describes the optimization of a uniform
                      procedure for the expression and purification of the wild
                      type NS4A(1-48) peptide and a peptide derived from a
                      replication-deficient mutant NS4A(1-48; L6E, M10E) with
                      disrupted amphipathic nature. A codon-optimized, synthetic
                      gene for NS4A(1-48) was expressed as a fusion with a GST-GB1
                      dual tag in E. coli. Tobacco etch virus (TEV) protease
                      mediated cleavage generated NS4A(1-48) peptides without any
                      artificial overhang. Using the described protocol up to 4
                      milligrams of the wild type or up to 5 milligrams of the
                      mutant peptide were obtained from a one-liter culture.
                      Isotopic labeling of the peptides was achieved and initial
                      NMR spectra were recorded.CONCLUSIONS/SIGNIFICANCE:Small
                      molecules targeting amphipathic helices in the related
                      Hepatitis C virus were shown to inhibit viral replication,
                      representing a new class of antiviral drugs. These findings
                      highlight the need for an efficient procedure that provides
                      large quantities of the amphipathic helix containing NS4A
                      peptides. The double tag strategy presented in this
                      manuscript answers these needs yielding amounts that are
                      sufficient for comprehensive biophysical and structural
                      studies, which might reveal new drug targets.},
      cin          = {ICS-6},
      ddc          = {500},
      cid          = {I:(DE-Juel1)ICS-6-20110106},
      pnm          = {452 - Structural Biology (POF2-452)},
      pid          = {G:(DE-HGF)POF2-452},
      typ          = {PUB:(DE-HGF)16},
      UT           = {WOS:000330288000105},
      pubmed       = {pmid:24466115},
      doi          = {10.1371/journal.pone.0086482},
      url          = {https://juser.fz-juelich.de/record/151099},
}