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Title
Expression, purification and crystallization of CTB-MPR, a candidate mucosal vaccine component against HIV-1
Description
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.
Date Created
2014-08-20
Contributors
- Lee, Ho-Hsien (Author)
- Cherni, Irene (Author)
- Yu, HongQi (Author)
- Fromme, Raimund (Author)
- Doran, Jeffrey (Author)
- Grotjohann, Ingo (Author)
- Mittman, Michele (Author)
- Basu, Shibom (Author)
- Deb, Arpan (Author)
- Dorner, Katerina (Author)
- Aquila, Andrew (Author)
- Barty, Anton (Author)
- Boutet, Sebastien (Author)
- Chapman, Henry N. (Author)
- Doak, R. Bruce (Author)
- Hunter, Mark (Author)
- James, Daniel (Author)
- Kirian, Richard (Author)
- Kupitz, Christopher (Author)
- Lawrence, Robert (Author)
- Liu, Haiguang (Author)
- Nass, Karol (Author)
- Schlichting, Ilme (Author)
- Schmidt, Kevin (Author)
- Seibert, M. Marvin (Author)
- Shoeman, Robert L. (Author)
- Spence, John (Author)
- Stellato, Francesco (Author)
- Weierstall, Uwe (Author)
- Williams, Garth J. (Author)
- Yoon, Chun Hong (Author)
- Wang, Dingjie (Author)
- Zatsepin, Nadia (Author)
- Hogue, Brenda (Author)
- Matoba, Nobuyuki (Author)
- Fromme, Petra (Author)
- Mor, Tsafrir (Author)
- ASU Biodesign Center Immunotherapy, Vaccines and Virotherapy (Contributor)
- Department of Chemistry and Biochemistry (Contributor)
- College of Liberal Arts and Sciences (Contributor)
- School of Life Sciences (Contributor)
- Biodesign Institute (Contributor)
- Infectious Diseases and Vaccinology (Contributor)
- Department of Physics (Contributor)
Resource Type
Extent
13 pages
Language
eng
Copyright Statement
In Copyright
Primary Member of
Identifier
Digital object identifier: 10.1107/S2052252514014900
Identifier Type
International standard serial number
Identifier Value
2052-2525
Series
IUCrJ
Handle
https://hdl.handle.net/2286/R.I.44820
Preferred Citation
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
Level of coding
minimal
Cataloging Standards
Note
View the article as published at http://journals.iucr.org/m/issues/2014/05/00/mf5003/index.html
System Created
- 2017-07-13 12:47:04
System Modified
- 2021-08-16 02:23:30
- 3 years 2 months ago
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