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Expression, purific...
Expression, purification and crystallization of CTB-MPR, a candidate mucosal vaccine component against HIV-1
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Lee, Ho-Hsien (author)
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Cherni, Irene (author)
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Yu, HongQi (author)
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show more...
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Fromme, Raimund (author)
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Doran, Jeffrey D. (author)
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Grotjohann, Ingo (author)
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Mittman, Michele (author)
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Basu, Shibom (author)
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Deb, Arpan (author)
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Dörner, Katerina (author)
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Aquila, Andrew (author)
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Barty, Anton (author)
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Boutet, Sébastien (author)
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Chapman, Henry N. (author)
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Doak, R. Bruce (author)
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Hunter, Mark S. (author)
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James, Daniel (author)
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Kirian, Richard A. (author)
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Kupitz, Christopher (author)
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Lawrence, Robert M. (author)
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Liu, Haiguang (author)
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Nass, Karol (author)
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Schlichting, Ilme (author)
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Schmidt, Kevin E. (author)
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- Seibert, M. Marvin (author)
- Linac Coherent Light Source, SLAC National Accelerator Laboratory, 2575 Sand Hill Road, Menlo Park, CA 94025, USA,
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Shoeman, Robert L. (author)
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Spence, John C. H. (author)
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Stellato, Francesco (author)
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Weierstall, Uwe (author)
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Williams, Garth J. (author)
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Yoon, Chunhong (author)
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Wang, Dingjie (author)
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Zatsepin, Nadia A. (author)
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Hogue, Brenda G. (author)
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Matoba, Nobuyuki (author)
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Fromme, Petra (author)
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Mor, Tsafrir S. (author)
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(creator_code:org_t)
- 2014
- 2014
- English.
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In: IUCrJ. - 2052-2525. ; 1:5, s. 305-317
- Related links:
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http://dx.doi.org/10...
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https://urn.kb.se/re...
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https://doi.org/10.1...
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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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- ref (subject category)
- art (subject category)
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IUCrJ
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- By the author/editor
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Lee, Ho-Hsien
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Cherni, Irene
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Yu, HongQi
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Fromme, Raimund
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Doran, Jeffrey D ...
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Grotjohann, Ingo
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show more...
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Mittman, Michele
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Basu, Shibom
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Deb, Arpan
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Dörner, Katerina
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Aquila, Andrew
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Barty, Anton
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Boutet, Sébastie ...
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Chapman, Henry N ...
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Doak, R. Bruce
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Hunter, Mark S.
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James, Daniel
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Kirian, Richard ...
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Kupitz, Christop ...
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Lawrence, Robert ...
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Liu, Haiguang
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Nass, Karol
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Schlichting, Ilm ...
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Schmidt, Kevin E ...
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Seibert, M. Marv ...
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Shoeman, Robert ...
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Spence, John C. ...
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Stellato, France ...
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Weierstall, Uwe
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Williams, Garth ...
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Yoon, Chunhong
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Wang, Dingjie
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Zatsepin, Nadia ...
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Hogue, Brenda G.
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Matoba, Nobuyuki
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Fromme, Petra
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Mor, Tsafrir S.
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show less...
- About the subject
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- NATURAL SCIENCES
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NATURAL SCIENCES
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and Biological Scien ...
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and Biochemistry and ...
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IUCrJ
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Uppsala University