title: Expression, purification and crystallization of CTB-MPR, a candidate mucosal vaccine component against HIV-1.
abstract: CTB-MPR is a fusion protein between the B subunit of cholera toxin (CTB) and the membrane-proximal region of gp41 (MPR), the transmembrane envelope protein of Human immunodeficiency virus 1 (HIV-1), and has previously been shown to induce the production of anti-HIV-1 antibodies with antiviral functions. To further improve the design of this candidate vaccine, X-ray crystallography experiments were performed to obtain structural information about this fusion protein. Several variants of CTB-MPR were designed, constructed and recombinantly expressed in Escherichia coli. The first variant contained a flexible GPGP linker between CTB and MPR, and yielded crystals that diffracted to a resolution of 2.3 Å, but only the CTB region was detected in the electron-density map. A second variant, in which the CTB was directly attached to MPR, was shown to destabilize pentamer formation. A third construct containing a polyalanine linker between CTB and MPR proved to stabilize the pentameric form of the protein during purification. The purification procedure was shown to produce a homogeneously pure and monodisperse sample for crystallization. Initial crystallization experiments led to pseudo-crystals which were ordered in only two dimensions and were disordered in the third dimension. Nanocrystals obtained using the same precipitant showed promising X-ray diffraction to 5 Å resolution in femtosecond nanocrystallography experiments at the Linac Coherent Light Source at the SLAC National Accelerator Laboratory. The results demonstrate the utility of femtosecond X-ray crystallography to enable structural analysis based on nano/microcrystals of a protein for which no macroscopic crystals ordered in three dimensions have been observed before.
authors.affiliation: Department of Chemistry and Biochemistry, Arizona State University , PO Box 871604, Tempe, AZ 85287-1604, USA.
authors.forename: Ho-Hsien
authors.initials: HH
authors.lastname: Lee
authors.affiliation: School of Life Sciences, Arizona State University , PO Box 874501, Tempe, AZ 85287-4501, USA ; Center for Infectious Diseases and Vaccinology, Biodesign Institute, Arizona State University , PO Box 874501, Tempe, AZ 85287-5401, USA.
authors.forename: Irene
authors.initials: I
authors.lastname: Cherni
authors.affiliation: Department of Chemistry and Biochemistry, Arizona State University , PO Box 871604, Tempe, AZ 85287-1604, USA.
authors.forename: HongQi
authors.initials: H
authors.lastname: Yu
authors.affiliation: Department of Chemistry and Biochemistry, Arizona State University , PO Box 871604, Tempe, AZ 85287-1604, USA.
authors.forename: Raimund
authors.initials: R
authors.lastname: Fromme
authors.affiliation: School of Life Sciences, Arizona State University , PO Box 874501, Tempe, AZ 85287-4501, USA ; Center for Infectious Diseases and Vaccinology, Biodesign Institute, Arizona State University , PO Box 874501, Tempe, AZ 85287-5401, USA.
authors.forename: Jeffrey D
authors.initials: JD
authors.lastname: Doran
authors.affiliation: Department of Chemistry and Biochemistry, Arizona State University , PO Box 871604, Tempe, AZ 85287-1604, USA.
authors.forename: Ingo
authors.initials: I
authors.lastname: Grotjohann
authors.affiliation: School of Life Sciences, Arizona State University , PO Box 874501, Tempe, AZ 85287-4501, USA ; Center for Infectious Diseases and Vaccinology, Biodesign Institute, Arizona State University , PO Box 874501, Tempe, AZ 85287-5401, USA.
authors.forename: Michele
authors.initials: M
authors.lastname: Mittman
authors.affiliation: Department of Chemistry and Biochemistry, Arizona State University , PO Box 871604, Tempe, AZ 85287-1604, USA.
authors.forename: Shibom
authors.initials: S
authors.lastname: Basu
authors.affiliation: School of Life Sciences, Arizona State University , PO Box 874501, Tempe, AZ 85287-4501, USA ; Center for Infectious Diseases and Vaccinology, Biodesign Institute, Arizona State University , PO Box 874501, Tempe, AZ 85287-5401, USA.
authors.forename: Arpan
authors.initials: A
authors.lastname: Deb
authors.affiliation: Department of Chemistry and Biochemistry, Arizona State University , PO Box 871604, Tempe, AZ 85287-1604, USA.
authors.forename: Katerina
authors.initials: K
authors.lastname: Dörner
authors.affiliation: Center for Free-Electron Laser Science, DESY , Notkestrasse 85, 22607 Hamburg, Germany.
authors.forename: Andrew
authors.initials: A
authors.lastname: Aquila
authors.affiliation: Center for Free-Electron Laser Science, DESY , Notkestrasse 85, 22607 Hamburg, Germany.
authors.forename: Anton
authors.initials: A
authors.lastname: Barty
authors.affiliation: Linac Coherent Light Source, SLAC National Accelerator Laboratory , 2575 Sand Hill Road, Menlo Park, CA 94025, USA.
authors.forename: Sébastien
authors.initials: S
authors.lastname: Boutet
authors.affiliation: Center for Free-Electron Laser Science, DESY , Notkestrasse 85, 22607 Hamburg, Germany ; University of Hamburg , Luruper Chaussee 149, 22761 Hamburg, Germany.
authors.forename: Henry N
authors.initials: HN
authors.lastname: Chapman
authors.affiliation: Department of Physics, Arizona State University , PO Box 871504, Tempe, AZ 85287-1504, USA.
authors.forename: R Bruce
authors.initials: RB
authors.lastname: Doak
authors.affiliation: Department of Chemistry and Biochemistry, Arizona State University , PO Box 871604, Tempe, AZ 85287-1604, USA.
authors.forename: Mark S
authors.initials: MS
authors.lastname: Hunter
authors.affiliation: Department of Physics, Arizona State University , PO Box 871504, Tempe, AZ 85287-1504, USA.
authors.forename: Daniel
authors.initials: D
authors.lastname: James
authors.affiliation: Center for Free-Electron Laser Science, DESY , Notkestrasse 85, 22607 Hamburg, Germany ; Department of Physics, Arizona State University , PO Box 871504, Tempe, AZ 85287-1504, USA.
authors.forename: Richard A
authors.initials: RA
authors.lastname: Kirian
authors.affiliation: Department of Chemistry and Biochemistry, Arizona State University , PO Box 871604, Tempe, AZ 85287-1604, USA.
authors.forename: Christopher
authors.initials: C
authors.lastname: Kupitz
authors.affiliation: Department of Chemistry and Biochemistry, Arizona State University , PO Box 871604, Tempe, AZ 85287-1604, USA ; Center for Infectious Diseases and Vaccinology, Biodesign Institute, Arizona State University , PO Box 874501, Tempe, AZ 85287-5401, USA.
authors.forename: Robert M
authors.initials: RM
authors.lastname: Lawrence
authors.affiliation: Department of Physics, Arizona State University , PO Box 871504, Tempe, AZ 85287-1504, USA.
authors.forename: Haiguang
authors.initials: H
authors.lastname: Liu
authors.affiliation: Center for Free-Electron Laser Science, DESY , Notkestrasse 85, 22607 Hamburg, Germany ; University of Hamburg , Luruper Chaussee 149, 22761 Hamburg, Germany.
authors.forename: Karol
authors.initials: K
authors.lastname: Nass
authors.affiliation: Max-Planck-Institut für medizinische Forschung , Jahnstrasse 29, 69120 Heidelberg, Germany.
authors.forename: Ilme
authors.initials: I
authors.lastname: Schlichting
authors.affiliation: Department of Physics, Arizona State University , PO Box 871504, Tempe, AZ 85287-1504, USA.
authors.forename: Kevin E
authors.initials: KE
authors.lastname: Schmidt
authors.affiliation: Linac Coherent Light Source, SLAC National Accelerator Laboratory , 2575 Sand Hill Road, Menlo Park, CA 94025, USA.
authors.forename: M Marvin
authors.initials: MM
authors.lastname: Seibert
authors.affiliation: Max-Planck-Institut für medizinische Forschung , Jahnstrasse 29, 69120 Heidelberg, Germany.
authors.forename: Robert L
authors.initials: RL
authors.lastname: Shoeman
authors.affiliation: Department of Physics, Arizona State University , PO Box 871504, Tempe, AZ 85287-1504, USA.
authors.forename: John C H
authors.initials: JC
authors.lastname: Spence
authors.affiliation: Center for Free-Electron Laser Science, DESY , Notkestrasse 85, 22607 Hamburg, Germany.
authors.forename: Francesco
authors.initials: F
authors.lastname: Stellato
authors.affiliation: Department of Physics, Arizona State University , PO Box 871504, Tempe, AZ 85287-1504, USA.
authors.forename: Uwe
authors.initials: U
authors.lastname: Weierstall
authors.affiliation: Linac Coherent Light Source, SLAC National Accelerator Laboratory , 2575 Sand Hill Road, Menlo Park, CA 94025, USA.
authors.forename: Garth J
authors.initials: GJ
authors.lastname: Williams
authors.affiliation: Center for Free-Electron Laser Science, DESY , Notkestrasse 85, 22607 Hamburg, Germany ; European XFEL GmbH , Albert-Einstein-Ring 19, 22761 Hamburg, Germany.
authors.forename: Chunhong
authors.initials: C
authors.lastname: Yoon
authors.affiliation: Department of Physics, Arizona State University , PO Box 871504, Tempe, AZ 85287-1504, USA.
authors.forename: Dingjie
authors.initials: D
authors.lastname: Wang
authors.affiliation: Department of Physics, Arizona State University , PO Box 871504, Tempe, AZ 85287-1504, USA.
authors.forename: Nadia A
authors.initials: NA
authors.lastname: Zatsepin
authors.affiliation: School of Life Sciences, Arizona State University , PO Box 874501, Tempe, AZ 85287-4501, USA ; Center for Infectious Diseases and Vaccinology, Biodesign Institute, Arizona State University , PO Box 874501, Tempe, AZ 85287-5401, USA.
authors.forename: Brenda G
authors.initials: BG
authors.lastname: Hogue
authors.affiliation: School of Life Sciences, Arizona State University , PO Box 874501, Tempe, AZ 85287-4501, USA ; Center for Infectious Diseases and Vaccinology, Biodesign Institute, Arizona State University , PO Box 874501, Tempe, AZ 85287-5401, USA.
authors.forename: Nobuyuki
authors.initials: N
authors.lastname: Matoba
authors.affiliation: Department of Chemistry and Biochemistry, Arizona State University , PO Box 871604, Tempe, AZ 85287-1604, USA.
authors.forename: Petra
authors.initials: P
authors.lastname: Fromme
authors.affiliation: School of Life Sciences, Arizona State University , PO Box 874501, Tempe, AZ 85287-4501, USA ; Center for Infectious Diseases and Vaccinology, Biodesign Institute, Arizona State University , PO Box 874501, Tempe, AZ 85287-5401, USA.
authors.forename: Tsafrir S
authors.initials: TS
authors.lastname: Mor
keywords: HIV-1; X-ray crystallography; cholera toxin B subunit; crystallization; femtosecond nanocrystallography; free-electron lasers; gp41; membrane-proximal region