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

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Fältnamn	Indikatorer	Metadata
000		04481naa a2200721 4500
001		oai:DiVA.org:uu-232888
003		SwePub
008		140926s2014 \| \|\|\|\|\|\|\|\|\|\|\|000 \|\|eng\|
024	7	a https://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-2328882 URI
024	7	a https://doi.org/10.1107/S20522525140149002 DOI
040		a (SwePub)uu
041		a engb eng
042		9 SwePub
072	7	a ref2 swepub-contenttype
072	7	a art2 swepub-publicationtype
100	1	a Lee, Ho-Hsien4 aut
245	1 0	a Expression, purification and crystallization of CTB-MPR, a candidate mucosal vaccine component against HIV-1
264	1	c 2014
338		a print2 rdacarrier
520		a 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.
650	7	a NATURVETENSKAPx Biologix Biokemi och molekylärbiologi0 (SwePub)106022 hsv//swe
650	7	a NATURAL SCIENCESx Biological Sciencesx Biochemistry and Molecular Biology0 (SwePub)106022 hsv//eng
700	1	a Cherni, Irene4 aut
700	1	a Yu, HongQi4 aut
700	1	a Fromme, Raimund4 aut
700	1	a Doran, Jeffrey D.4 aut
700	1	a Grotjohann, Ingo4 aut
700	1	a Mittman, Michele4 aut
700	1	a Basu, Shibom4 aut
700	1	a Deb, Arpan4 aut
700	1	a Dörner, Katerina4 aut
700	1	a Aquila, Andrew4 aut
700	1	a Barty, Anton4 aut
700	1	a Boutet, Sébastien4 aut
700	1	a Chapman, Henry N.4 aut
700	1	a Doak, R. Bruce4 aut
700	1	a Hunter, Mark S.4 aut
700	1	a James, Daniel4 aut
700	1	a Kirian, Richard A.4 aut
700	1	a Kupitz, Christopher4 aut
700	1	a Lawrence, Robert M.4 aut
700	1	a Liu, Haiguang4 aut
700	1	a Nass, Karol4 aut
700	1	a Schlichting, Ilme4 aut
700	1	a Schmidt, Kevin E.4 aut
700	1	a Seibert, M. Marvinu Linac Coherent Light Source, SLAC National Accelerator Laboratory, 2575 Sand Hill Road, Menlo Park, CA 94025, USA,4 aut0 (Swepub:uu)masei255
700	1	a Shoeman, Robert L.4 aut
700	1	a Spence, John C. H.4 aut
700	1	a Stellato, Francesco4 aut
700	1	a Weierstall, Uwe4 aut
700	1	a Williams, Garth J.4 aut
700	1	a Yoon, Chunhong4 aut
700	1	a Wang, Dingjie4 aut
700	1	a Zatsepin, Nadia A.4 aut
700	1	a Hogue, Brenda G.4 aut
700	1	a Matoba, Nobuyuki4 aut
700	1	a Fromme, Petra4 aut
700	1	a Mor, Tsafrir S.4 aut
710	2	a Linac Coherent Light Source, SLAC National Accelerator Laboratory, 2575 Sand Hill Road, Menlo Park, CA 94025, USA,4 org
773	0	t IUCrJg 1:5, s. 305-317q 1:5<305-317x 2052-2525
856	4	u http://dx.doi.org/10.1107/S2052252514014900
856	4 8	u https://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-232888
856	4 8	u https://doi.org/10.1107/S2052252514014900

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