Description
CTB-MPR is a fusion protein between the B subunit of cholera toxin (CTB) and the membrane-proximal region of gp41 (MPR), the transmembrane envelope protein of Human immunodeficiency virus 1 (HIV-1), and has previously been shown to induce the production of

CTB-MPR is a fusion protein between the B subunit of cholera toxin (CTB) and the membrane-proximal region of gp41 (MPR), the transmembrane envelope protein of Human immunodeficiency virus 1 (HIV-1), and has previously been shown to induce the production of anti-HIV-1 antibodies with antiviral functions. To further improve the design of this candidate vaccine, X-ray crystallography experiments were performed to obtain structural information about this fusion protein. Several variants of CTB-MPR were designed, constructed and recombinantly expressed in Escherichia coli. The first variant contained a flexible GPGP linker between CTB and MPR, and yielded crystals that diffracted to a resolution of 2.3 Å, but only the CTB region was detected in the electron-density map. A second variant, in which the CTB was directly attached to MPR, was shown to destabilize pentamer formation. A third construct containing a polyalanine linker between CTB and MPR proved to stabilize the pentameric form of the protein during purification. The purification procedure was shown to produce a homogeneously pure and monodisperse sample for crystallization. Initial crystallization experiments led to pseudo-crystals which were ordered in only two dimensions and were disordered in the third dimension. Nanocrystals obtained using the same precipitant showed promising X-ray diffraction to 5 Å resolution in femtosecond nanocrystallography experiments at the Linac Coherent Light Source at the SLAC National Accelerator Laboratory. The results demonstrate the utility of femtosecond X-ray crystallography to enable structural analysis based on nano/microcrystals of a protein for which no macroscopic crystals ordered in three dimensions have been observed before.
Reuse Permissions
  • Downloads
    PDF (1.8 MB)

    Details

    Title
    • Expression, purification and crystallization of CTB-MPR, a candidate mucosal vaccine component against HIV-1
    Date Created
    2014-08-20
    Resource Type
  • Text
  • Collections this item is in
    Identifier
    • Digital object identifier: 10.1107/S2052252514014900
    • Identifier Type
      International standard serial number
      Identifier Value
      2052-2525
    Note
    • View the article as published at http://journals.iucr.org/m/issues/2014/05/00/mf5003/index.html

    Citation and reuse

    Cite this item

    This is a suggested citation. Consult the appropriate style guide for specific citation guidelines.

    Lee, H., Cherni, I., Yu, H., Fromme, R., Doran, J. D., Grotjohann, I., . . . Mor, T. S. (2014). Expression, purification and crystallization of CTB-MPR, a candidate mucosal vaccine component against HIV-1. IUCrJ, 1(5), 305-317. doi:10.1107/s2052252514014900

    Machine-readable links