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

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

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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 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.

Original languageEnglish
Pages (from-to)305-317
Number of pages13
JournalIUCrJ
Volume1
DOIs
Publication statusPublished - Aug 29 2014

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Keywords

  • HIV-1
  • X-ray crystallography
  • cholera toxin B subunit
  • crystallization
  • femtosecond nanocrystallography
  • free-electron lasers
  • gp41
  • membrane-proximal region

ASJC Scopus subject areas

  • Chemistry(all)
  • Biochemistry
  • Materials Science(all)
  • Condensed Matter Physics

Cite this

Lee, H. H., Cherni, I., Yu, H., Fromme, R., Doran, J. D., Grotjohann, I., Mittman, M., Basu, S., Deb, A., Dörner, K., Aquila, A., Barty, A., Boutet, S., Chapman, H. N., Doak, R. B., Hunter, M. S., James, D., Kirian, R. A., Kupitz, C., ... Mor, T. S. (2014). Expression, purification and crystallization of CTB-MPR, a candidate mucosal vaccine component against HIV-1. IUCrJ, 1, 305-317. https://doi.org/10.1107/S2052252514014900