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Expression, purific...
Expression, purification and crystallization of CTB-MPR, a candidate mucosal vaccine component against HIV-1
- Article/chapterEnglish2014
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Numbers
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LIBRIS-ID:oai:DiVA.org:uu-232888
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https://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-232888URI
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https://doi.org/10.1107/S2052252514014900DOI
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Language:English
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Summary in:English
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Subject category:ref swepub-contenttype
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Subject category:art swepub-publicationtype
Notes
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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 and genre
Added entries (persons, corporate bodies, meetings, titles ...)
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Cherni, Irene
(author)
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Yu, HongQi
(author)
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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. MarvinLinac Coherent Light Source, SLAC National Accelerator Laboratory, 2575 Sand Hill Road, Menlo Park, CA 94025, USA,(Swepub:uu)masei255
(author)
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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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Linac Coherent Light Source, SLAC National Accelerator Laboratory, 2575 Sand Hill Road, Menlo Park, CA 94025, USA,
(creator_code:org_t)
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In:IUCrJ1:5, s. 305-3172052-2525
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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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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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