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Expression, purification and crystallization of CTB-MPR, a candidate mucosal vaccine component against HIV-1
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2014 (English)In: IUCrJ, ISSN 2052-2525, Vol. 1, no 5, 305-317 p.Article in journal (Refereed) Published
Abstract [en]

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.

Place, publisher, year, edition, pages
2014. Vol. 1, no 5, 305-317 p.
National Category
Biochemistry and Molecular Biology
URN: urn:nbn:se:uu:diva-232888DOI: 10.1107/S2052252514014900OAI: oai:DiVA.org:uu-232888DiVA: diva2:750104
Available from: 2014-09-26 Created: 2014-09-26 Last updated: 2014-10-01Bibliographically approved

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Publisher's full texthttp://dx.doi.org/10.1107/S2052252514014900

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Seibert, M. Marvin
Biochemistry and Molecular Biology

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