Expression, purification and crystallization of CTB-MPR, a candidate mucosal vaccine component against HIV-1
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 functi...
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International Union of Crystallography
2014-09-01
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Online Access: | http://scripts.iucr.org/cgi-bin/paper?S2052252514014900 |
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Article |
collection |
DOAJ |
language |
English |
format |
Article |
sources |
DOAJ |
author |
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 |
spellingShingle |
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 Expression, purification and crystallization of CTB-MPR, a candidate mucosal vaccine component against HIV-1 IUCrJ X-ray crystallography femtosecond nanocrystallography HIV-1 gp41 membrane-proximal region cholera toxin B subunit crystallization free-electron lasers |
author_facet |
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 |
author_sort |
Ho-Hsien Lee |
title |
Expression, purification and crystallization of CTB-MPR, a candidate mucosal vaccine component against HIV-1 |
title_short |
Expression, purification and crystallization of CTB-MPR, a candidate mucosal vaccine component against HIV-1 |
title_full |
Expression, purification and crystallization of CTB-MPR, a candidate mucosal vaccine component against HIV-1 |
title_fullStr |
Expression, purification and crystallization of CTB-MPR, a candidate mucosal vaccine component against HIV-1 |
title_full_unstemmed |
Expression, purification and crystallization of CTB-MPR, a candidate mucosal vaccine component against HIV-1 |
title_sort |
expression, purification and crystallization of ctb-mpr, a candidate mucosal vaccine component against hiv-1 |
publisher |
International Union of Crystallography |
series |
IUCrJ |
issn |
2052-2525 |
publishDate |
2014-09-01 |
description |
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. |
topic |
X-ray crystallography femtosecond nanocrystallography HIV-1 gp41 membrane-proximal region cholera toxin B subunit crystallization free-electron lasers |
url |
http://scripts.iucr.org/cgi-bin/paper?S2052252514014900 |
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doaj-e93878e8686c49afaf11b3e9c588d63b2020-11-24T21:59:50ZengInternational Union of CrystallographyIUCrJ2052-25252014-09-011530531710.1107/S2052252514014900mf5003Expression, purification and crystallization of CTB-MPR, a candidate mucosal vaccine component against HIV-1Ho-Hsien Lee0Irene Cherni1HongQi Yu2Raimund Fromme3Jeffrey D. Doran4Ingo Grotjohann5Michele Mittman6Shibom Basu7Arpan Deb8Katerina Dörner9Andrew Aquila10Anton Barty11Sébastien Boutet12Henry N. Chapman13R. Bruce Doak14Mark S. Hunter15Daniel James16Richard A. Kirian17Christopher Kupitz18Robert M. Lawrence19Haiguang Liu20Karol Nass21Ilme Schlichting22Kevin E. Schmidt23M. Marvin Seibert24Robert L. Shoeman25John C. H. Spence26Francesco Stellato27Uwe Weierstall28Garth J. Williams29Chunhong Yoon30Dingjie Wang31Nadia A. Zatsepin32Brenda G. Hogue33Nobuyuki Matoba34Petra Fromme35Tsafrir S. Mor36Department of Chemistry and Biochemistry, Arizona State University, PO Box 871604, Tempe, AZ 85287-1604, USASchool of Life Sciences, Arizona State University, PO Box 874501, Tempe, AZ 85287-4501, USADepartment of Chemistry and Biochemistry, Arizona State University, PO Box 871604, Tempe, AZ 85287-1604, USADepartment of Chemistry and Biochemistry, Arizona State University, PO Box 871604, Tempe, AZ 85287-1604, USASchool of Life Sciences, Arizona State University, PO Box 874501, Tempe, AZ 85287-4501, USADepartment of Chemistry and Biochemistry, Arizona State University, PO Box 871604, Tempe, AZ 85287-1604, USASchool of Life Sciences, Arizona State University, PO Box 874501, Tempe, AZ 85287-4501, USADepartment of Chemistry and Biochemistry, Arizona State University, PO Box 871604, Tempe, AZ 85287-1604, USASchool of Life Sciences, Arizona State University, PO Box 874501, Tempe, AZ 85287-4501, USADepartment of Chemistry and Biochemistry, Arizona State University, PO Box 871604, Tempe, AZ 85287-1604, USACenter for Free-Electron Laser Science, DESY, Notkestrasse 85, 22607 Hamburg, GermanyCenter for Free-Electron Laser Science, DESY, Notkestrasse 85, 22607 Hamburg, GermanyLinac Coherent Light Source, SLAC National Accelerator Laboratory, 2575 Sand Hill Road, Menlo Park, CA 94025, USACenter for Free-Electron Laser Science, DESY, Notkestrasse 85, 22607 Hamburg, GermanyDepartment of Physics, Arizona State University, PO Box 871504, Tempe, AZ 85287-1504, USADepartment of Chemistry and Biochemistry, Arizona State University, PO Box 871604, Tempe, AZ 85287-1604, USADepartment of Physics, Arizona State University, PO Box 871504, Tempe, AZ 85287-1504, USACenter for Free-Electron Laser Science, DESY, Notkestrasse 85, 22607 Hamburg, GermanyDepartment of Chemistry and Biochemistry, Arizona State University, PO Box 871604, Tempe, AZ 85287-1604, USADepartment of Chemistry and Biochemistry, Arizona State University, PO Box 871604, Tempe, AZ 85287-1604, USADepartment of Physics, Arizona State University, PO Box 871504, Tempe, AZ 85287-1504, USACenter for Free-Electron Laser Science, DESY, Notkestrasse 85, 22607 Hamburg, GermanyMax-Planck-Institut für medizinische Forschung, Jahnstrasse 29, 69120 Heidelberg, GermanyDepartment of Physics, Arizona State University, PO Box 871504, Tempe, AZ 85287-1504, USALinac Coherent Light Source, SLAC National Accelerator Laboratory, 2575 Sand Hill Road, Menlo Park, CA 94025, USAMax-Planck-Institut für medizinische Forschung, Jahnstrasse 29, 69120 Heidelberg, GermanyDepartment of Physics, Arizona State University, PO Box 871504, Tempe, AZ 85287-1504, USACenter for Free-Electron Laser Science, DESY, Notkestrasse 85, 22607 Hamburg, GermanyDepartment of Physics, Arizona State University, PO Box 871504, Tempe, AZ 85287-1504, USALinac Coherent Light Source, SLAC National Accelerator Laboratory, 2575 Sand Hill Road, Menlo Park, CA 94025, USACenter for Free-Electron Laser Science, DESY, Notkestrasse 85, 22607 Hamburg, GermanyDepartment of Physics, Arizona State University, PO Box 871504, Tempe, AZ 85287-1504, USADepartment of Physics, Arizona State University, PO Box 871504, Tempe, AZ 85287-1504, USASchool of Life Sciences, Arizona State University, PO Box 874501, Tempe, AZ 85287-4501, USASchool of Life Sciences, Arizona State University, PO Box 874501, Tempe, AZ 85287-4501, USADepartment of Chemistry and Biochemistry, Arizona State University, PO Box 871604, Tempe, AZ 85287-1604, USASchool of Life Sciences, Arizona State University, PO Box 874501, Tempe, AZ 85287-4501, USACTB-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.http://scripts.iucr.org/cgi-bin/paper?S2052252514014900X-ray crystallographyfemtosecond nanocrystallographyHIV-1gp41membrane-proximal regioncholera toxin B subunitcrystallizationfree-electron lasers |