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130284 https://hdl.handle.net/2286/R.I.44820 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 10.1107/S2052252514014900 2052-2525 http://rightsstatements.org/vocab/InC/1.0/ http://creativecommons.org/licenses/by/4.0 2014-08-20 13 pages eng Lee, Ho-Hsien Cherni, Irene Yu, HongQi Fromme, Raimund Doran, Jeffrey Grotjohann, Ingo Mittman, Michele Basu, Shibom Deb, Arpan Dorner, Katerina Aquila, Andrew Barty, Anton Boutet, Sebastien Chapman, Henry N. Doak, R. Bruce Hunter, Mark James, Daniel Kirian, Richard Kupitz, Christopher Lawrence, Robert Liu, Haiguang Nass, Karol Schlichting, Ilme Schmidt, Kevin Seibert, M. Marvin Shoeman, Robert L. Spence, John Stellato, Francesco Weierstall, Uwe Williams, Garth J. Yoon, Chun Hong Wang, Dingjie Zatsepin, Nadia Hogue, Brenda Matoba, Nobuyuki Fromme, Petra Mor, Tsafrir ASU Biodesign Center Immunotherapy, Vaccines and Virotherapy Department of Chemistry and Biochemistry College of Liberal Arts and Sciences School of Life Sciences Biodesign Institute Infectious Diseases and Vaccinology Department of Physics View the article as published at http://journals.iucr.org/m/issues/2014/05/00/mf5003/index.html 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. Text Expression, purification and crystallization of CTB-MPR, a candidate mucosal vaccine component against HIV-1