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

Lee, Ho-Hsien (author)
Cherni, Irene (author)
Yu, HongQi (author)
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Fromme, Raimund (author)
Doran, Jeffrey D. (author)
Grotjohann, Ingo (author)
Mittman, Michele (author)
Basu, Shibom (author)
Deb, Arpan (author)
Dörner, Katerina (author)
Aquila, Andrew (author)
Barty, Anton (author)
Boutet, Sébastien (author)
Chapman, Henry N. (author)
Doak, R. Bruce (author)
Hunter, Mark S. (author)
James, Daniel (author)
Kirian, Richard A. (author)
Kupitz, Christopher (author)
Lawrence, Robert M. (author)
Liu, Haiguang (author)
Nass, Karol (author)
Schlichting, Ilme (author)
Schmidt, Kevin E. (author)
Seibert, M. Marvin (author)
Linac Coherent Light Source, SLAC National Accelerator Laboratory, 2575 Sand Hill Road, Menlo Park, CA 94025, USA,
Shoeman, Robert L. (author)
Spence, John C. H. (author)
Stellato, Francesco (author)
Weierstall, Uwe (author)
Williams, Garth J. (author)
Yoon, Chunhong (author)
Wang, Dingjie (author)
Zatsepin, Nadia A. (author)
Hogue, Brenda G. (author)
Matoba, Nobuyuki (author)
Fromme, Petra (author)
Mor, Tsafrir S. (author)
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 (creator_code:org_t)
2014
2014
English.
In: IUCrJ. - 2052-2525. ; 1:5, s. 305-317
Related links:
http://dx.doi.org/10...
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https://urn.kb.se/re...
https://doi.org/10.1...
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  • Journal article (peer-reviewed)
Abstract Subject headings
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  • CTB-MPR is a fusion protein between the B subunit of cholera toxin (CTB) andthe membrane-proximal region of gp41 (MPR), the transmembrane envelopeprotein ofHuman immunodeficiency virus 1(HIV-1), and has previously beenshown to induce the production of anti-HIV-1 antibodies with antiviralfunctions. To further improve the design of this candidate vaccine, X-raycrystallography experiments were performed to obtain structural informationabout this fusion protein. Several variants of CTB-MPR were designed,constructed and recombinantly expressed inEscherichia coli. The first variantcontained a flexible GPGP linker between CTB and MPR, and yielded crystalsthat diffracted to a resolution of 2.3 A ̊, but only the CTB region was detectedin the electron-density map. A second variant, in which the CTB was directlyattached to MPR, was shown to destabilize pentamer formation. A thirdconstruct containing a polyalanine linker between CTB and MPR proved tostabilize the pentameric form of the protein during purification. The purificationprocedure was shown to produce a homogeneously pure and monodispersesample for crystallization. Initial crystallization experiments led to pseudo-crystals which were ordered in only two dimensions and were disordered inthe third dimension. Nanocrystals obtained using the same precipitant showedpromising X-ray diffraction to 5 A ̊resolution in femtosecond nanocrystallo-graphy experiments at the Linac Coherent Light Source at the SLAC NationalAccelerator Laboratory. The results demonstrate the utility of femtosecondX-ray crystallography to enable structural analysis based on nano/microcrystalsof a protein for which no macroscopic crystals ordered in three dimensions havebeen observed before.

Subject headings

NATURVETENSKAP  -- Biologi -- Biokemi och molekylärbiologi (hsv//swe)
NATURAL SCIENCES  -- Biological Sciences -- Biochemistry and Molecular Biology (hsv//eng)

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