Expression, purification and crystallization of CTB-MPR, a candidate mucosal vaccine component against HIV-1 Academic Article uri icon

abstract

  • 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 ofHuman 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 inEscherichia 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.

authors

  • Lee, Ho-Hsien
  • Cherni, Irene
  • Yu, HongQi
  • Fromme, Raimund
  • Doran, Jeffrey D
  • Grotjohann, Ingo
  • Mittman, Michele
  • Basu, Shibom
  • Deb, Arpan
  • Dörner, Katerina
  • Aquila, Andrew
  • Barty, Anton
  • Boutet, Sébastien
  • Chapman, Henry N
  • Doak, R Bruce
  • Hunter, Mark S
  • James, Daniel
  • Kirian, Richard A
  • Kupitz, Christopher
  • Lawrence, Robert M
  • Liu, Haiguang
  • Nass, Karol
  • Schlichting, Ilme
  • Schmidt, Kevin E
  • Seibert, M Marvin
  • Shoeman, Robert L
  • Spence, John CH
  • Stellato, Francesco
  • Weierstall, Uwe
  • Williams, Garth J
  • Yoon, Chunhong
  • Wang, Dingjie
  • Zatsepin, Nadia A
  • Hogue, Brenda G
  • Matoba, Nobuyuki
  • Fromme, Petra
  • Mor, Tsafrir S

publication date

  • 2014

published in