Expression, purification and crystallization of CTB-MPR, a candidate mucosal vaccine component against HIV-1
Category
Published on
Type
journal-article
Author
Ho-Hsien Lee and Irene Cherni and HongQi Yu and Raimund Fromme and Jeffrey D. Doran and Ingo Grotjohann and Michele Mittman and Shibom Basu and Arpan Deb and Katerina Dörner and Andrew Aquila and Anton Barty and Sébastien Boutet and Henry N. Chapman and R. Bruce Doak and Mark S. Hunter and Daniel James and Richard A. Kirian and Christopher Kupitz and Robert M. Lawrence and Haiguang Liu and Karol Nass and Ilme Schlichting and Kevin E. Schmidt and M. Marvin Seibert and Robert L. Shoeman and John C. H. Spence and Francesco Stellato and Uwe Weierstall and Garth J. Williams and Chunhong Yoon and Dingjie Wang and Nadia A. Zatsepin and Brenda G. Hogue and Nobuyuki Matoba and Petra Fromme and Tsafrir S. Mor
Citation
Lee, H.-H. et al., 2014. Expression, purification and crystallization of CTB-MPR, a candidate mucosal vaccine component against HIV-1. IUCrJ, 1(5), pp.305–317. Available at: http://dx.doi.org/10.1107/s2052252514014900.
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.
