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

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 tomore » 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.« less

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. more »; Liu, Haiguang; Nass, Karol; Schlichting, Ilme; Schmidt, Kevin E.; Seibert, M. Marvin; Shoeman, Robert L.; Spence, John C. H.; Stellato, Francesco; Weierstall, Uwe; Williams, Garth J.; Yoon, Chunhong; Wang, Dingjie; Zatsepin, Nadia A.; Hogue, Brenda G.; Matoba, Nobuyuki; Fromme, Petra; Mor, Tsafrir S. « less

Publication Date:

Wed Aug 20 00:00:00 EDT 2014

Sponsoring Org.:

USDOE

OSTI Identifier:

1335054

Resource Type:

Published Article

Journal Name:

IUCrJ

Additional Journal Information:

Journal Name: IUCrJ Journal Volume: 1 Journal Issue: 5; Journal ID: ISSN 2052-2525

Publisher:

International Union of Crystallography (IUCr)

Country of Publication:

United Kingdom

Language:

English