IUCrJ (Sep 2014)
Expression, purification and crystallization of CTB-MPR, a candidate mucosal vaccine component against HIV-1
- Ho-Hsien Lee,
- Irene Cherni,
- HongQi Yu,
- Raimund Fromme,
- Jeffrey D. Doran,
- Ingo Grotjohann,
- Michele Mittman,
- Shibom Basu,
- Arpan Deb,
- Katerina Dörner,
- Andrew Aquila,
- Anton Barty,
- Sébastien Boutet,
- Henry N. Chapman,
- R. Bruce Doak,
- Mark S. Hunter,
- Daniel James,
- Richard A. Kirian,
- Christopher Kupitz,
- Robert M. Lawrence,
- Haiguang Liu,
- Karol Nass,
- Ilme Schlichting,
- Kevin E. Schmidt,
- M. Marvin Seibert,
- Robert L. Shoeman,
- John C. H. Spence,
- Francesco Stellato,
- Uwe Weierstall,
- Garth J. Williams,
- Chunhong Yoon,
- Dingjie Wang,
- Nadia A. Zatsepin,
- Brenda G. Hogue,
- Nobuyuki Matoba,
- Petra Fromme,
- Tsafrir S. Mor
Affiliations
- Ho-Hsien Lee
- Department of Chemistry and Biochemistry, Arizona State University, PO Box 871604, Tempe, AZ 85287-1604, USA
- Irene Cherni
- School of Life Sciences, Arizona State University, PO Box 874501, Tempe, AZ 85287-4501, USA
- HongQi Yu
- Department of Chemistry and Biochemistry, Arizona State University, PO Box 871604, Tempe, AZ 85287-1604, USA
- Raimund Fromme
- Department of Chemistry and Biochemistry, Arizona State University, PO Box 871604, Tempe, AZ 85287-1604, USA
- Jeffrey D. Doran
- School of Life Sciences, Arizona State University, PO Box 874501, Tempe, AZ 85287-4501, USA
- Ingo Grotjohann
- Department of Chemistry and Biochemistry, Arizona State University, PO Box 871604, Tempe, AZ 85287-1604, USA
- Michele Mittman
- School of Life Sciences, Arizona State University, PO Box 874501, Tempe, AZ 85287-4501, USA
- Shibom Basu
- Department of Chemistry and Biochemistry, Arizona State University, PO Box 871604, Tempe, AZ 85287-1604, USA
- Arpan Deb
- School of Life Sciences, Arizona State University, PO Box 874501, Tempe, AZ 85287-4501, USA
- Katerina Dörner
- Department of Chemistry and Biochemistry, Arizona State University, PO Box 871604, Tempe, AZ 85287-1604, USA
- Andrew Aquila
- Center for Free-Electron Laser Science, DESY, Notkestrasse 85, 22607 Hamburg, Germany
- Anton Barty
- Center for Free-Electron Laser Science, DESY, Notkestrasse 85, 22607 Hamburg, Germany
- Sébastien Boutet
- Linac Coherent Light Source, SLAC National Accelerator Laboratory, 2575 Sand Hill Road, Menlo Park, CA 94025, USA
- Henry N. Chapman
- Center for Free-Electron Laser Science, DESY, Notkestrasse 85, 22607 Hamburg, Germany
- R. Bruce Doak
- Department of Physics, Arizona State University, PO Box 871504, Tempe, AZ 85287-1504, USA
- Mark S. Hunter
- Department of Chemistry and Biochemistry, Arizona State University, PO Box 871604, Tempe, AZ 85287-1604, USA
- Daniel James
- Department of Physics, Arizona State University, PO Box 871504, Tempe, AZ 85287-1504, USA
- Richard A. Kirian
- Center for Free-Electron Laser Science, DESY, Notkestrasse 85, 22607 Hamburg, Germany
- Christopher Kupitz
- Department of Chemistry and Biochemistry, Arizona State University, PO Box 871604, Tempe, AZ 85287-1604, USA
- Robert M. Lawrence
- Department of Chemistry and Biochemistry, Arizona State University, PO Box 871604, Tempe, AZ 85287-1604, USA
- Haiguang Liu
- Department of Physics, Arizona State University, PO Box 871504, Tempe, AZ 85287-1504, USA
- Karol Nass
- Center for Free-Electron Laser Science, DESY, Notkestrasse 85, 22607 Hamburg, Germany
- Ilme Schlichting
- Max-Planck-Institut für medizinische Forschung, Jahnstrasse 29, 69120 Heidelberg, Germany
- Kevin E. Schmidt
- Department of Physics, Arizona State University, PO Box 871504, Tempe, AZ 85287-1504, USA
- M. Marvin Seibert
- Linac Coherent Light Source, SLAC National Accelerator Laboratory, 2575 Sand Hill Road, Menlo Park, CA 94025, USA
- Robert L. Shoeman
- Max-Planck-Institut für medizinische Forschung, Jahnstrasse 29, 69120 Heidelberg, Germany
- John C. H. Spence
- Department of Physics, Arizona State University, PO Box 871504, Tempe, AZ 85287-1504, USA
- Francesco Stellato
- Center for Free-Electron Laser Science, DESY, Notkestrasse 85, 22607 Hamburg, Germany
- Uwe Weierstall
- Department of Physics, Arizona State University, PO Box 871504, Tempe, AZ 85287-1504, USA
- Garth J. Williams
- Linac Coherent Light Source, SLAC National Accelerator Laboratory, 2575 Sand Hill Road, Menlo Park, CA 94025, USA
- Chunhong Yoon
- Center for Free-Electron Laser Science, DESY, Notkestrasse 85, 22607 Hamburg, Germany
- Dingjie Wang
- Department of Physics, Arizona State University, PO Box 871504, Tempe, AZ 85287-1504, USA
- Nadia A. Zatsepin
- Department of Physics, Arizona State University, PO Box 871504, Tempe, AZ 85287-1504, USA
- Brenda G. Hogue
- School of Life Sciences, Arizona State University, PO Box 874501, Tempe, AZ 85287-4501, USA
- Nobuyuki Matoba
- School of Life Sciences, Arizona State University, PO Box 874501, Tempe, AZ 85287-4501, USA
- Petra Fromme
- Department of Chemistry and Biochemistry, Arizona State University, PO Box 871604, Tempe, AZ 85287-1604, USA
- Tsafrir S. Mor
- School of Life Sciences, Arizona State University, PO Box 874501, Tempe, AZ 85287-4501, USA
- DOI
- https://doi.org/10.1107/S2052252514014900
- Journal volume & issue
-
Vol. 1,
no. 5
pp. 305 – 317
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.
Keywords
- X-ray crystallography
- femtosecond nanocrystallography
- HIV-1
- gp41
- membrane-proximal region
- cholera toxin B subunit
- crystallization
- free-electron lasers
