Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2012 Jul 1;68(Pt 7):778-82.
doi: 10.1107/S1744309112019306. Epub 2012 Jun 27.

Expression, purification, crystallization and preliminary X-ray crystallographic analysis of Enpp1

Kazuki Kato  1 , Hiroshi Nishimasu, Emiko Mihara, Ryuichiro Ishitani, Junichi Takagi, Junken Aoki, Osamu Nureki
Affiliations

Expression, purification, crystallization and preliminary X-ray crystallographic analysis of Enpp1

Kazuki Kato et al. Acta Crystallogr Sect F Struct Biol Cryst Commun. .

Abstract

Enpp1 is an extracellular membrane-bound glycoprotein that regulates bone mineralization by hydrolyzing ATP to generate pyrophosphate. The extracellular region of mouse Enpp1 was expressed in HEK293S GnT1(-) cells, purified using the TARGET tag/P20.1-Sepharose system and crystallized. An X-ray diffraction data set was collected to 3.0 Å resolution. The crystal belonged to space group P3(1), with unit-cell parameters a = b = 105.3, c = 173.7 Å. A single-wavelength anomalous dispersion (SAD) data set was also collected to 2.7 Å resolution using a selenomethionine-labelled crystal. The experimental phases determined by the SAD method produced an interpretable electron-density map.

PubMed Disclaimer

Figures

Figure 1
Figure 1
Preparation of the extracellular region of Enpp1. (a) Construction of Enpp1. The domain organizations of Enpp1 and Enpp2 and a sequence alignment of the N-terminal regions of their SMB1 domains are shown. (b) Purification of Enpp1. Proteins were analyzed by SDS–PAGE and stained with SimplyBlue SafeStain. Lane 1, molecular-weight markers (labelled in kDa); lane 2, culture supernatant; lane 3, C-terminally tagged Enpp1 protein after P20.1-Sepharose chromatography; lane 4, purified Enpp1 protein after cleavage of the TARGET tag and gel-filtration chromatography.
Figure 2
Figure 2
Crystals of Enpp1. (a) Native crystals obtained under the initial conditions. (b) Native crystals obtained under the refined conditions. (c) SeMet-labelled crystals obtained under the refined conditions. Scale bars indicate 100 µm.
Figure 3
Figure 3
X-ray diffraction patterns of Enpp1. (a) Diffraction pattern of the native crystal. (b) Diffraction pattern of the SeMet-labelled crystal.
See this image and copyright information in PMC

References

    1. Adams, P. D., Grosse-Kunstleve, R. W., Hung, L.-W., Ioerger, T. R., McCoy, A. J., Moriarty, N. W., Read, R. J., Sacchettini, J. C., Sauter, N. K. & Terwilliger, T. C. (2002). Acta Cryst. D58, 1948–1954. - PubMed
    1. Emsley, P. & Cowtan, K. (2004). Acta Cryst. D60, 2126–2132. - PubMed
    1. Hamdan, S., Bulloch, E. M., Thompson, P. R., Beck, J. L., Yang, J. Y., Crowther, J. A., Lilley, P. E., Carr, P. D., Ollis, D. L., Brown, S. E. & Dixon, N. E. (2002). Biochemistry, 41, 5266–5275. - PubMed
    1. Hausmann, J. et al. (2011). Nature Struct. Mol. Biol. 18, 198–204. - PMC - PubMed
    1. Hessle, L., Johnson, K. A., Anderson, H. C., Narisawa, S., Sali, A., Goding, J. W., Terkeltaub, R. & Millan, J. L. (2002). Proc. Natl Acad. Sci. USA, 99, 9445–9449. - PMC - PubMed

Publication types

Substances