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. 2013 Oct;69(Pt 10):1155-8.
doi: 10.1107/S1744309113024330. Epub 2013 Sep 28.

Expression, purification and preliminary X-ray crystallographic analysis of Arf1-GDP in complex with dimeric p23 peptide

Peng Zheng  1 , Feng Gao, Kai Deng, Weimin Gong, Zhe Sun
Affiliations

Expression, purification and preliminary X-ray crystallographic analysis of Arf1-GDP in complex with dimeric p23 peptide

Peng Zheng et al. Acta Crystallogr Sect F Struct Biol Cryst Commun. 2013 Oct.

Abstract

Arf1 is a member of the Ras superfamily and is involved in COPI vesicle formation. Arf1-GDP can interact with dimeric p23. Here, human Arf1 (residues 18-181) was cloned, expressed and purified in Escherichia coli. For crystallization, Arf1-GDP was mixed with dimeric p23 peptide in a 1:5 molar ratio. Crystals were obtained which diffracted to 2.7 Å resolution. The crystals belonged to space group P6₁22, with unit-cell parameters a=b=80.6, c=336.0 Å, α=β=90, γ=120°. The asymmetric unit of the crystals contained two molecules, with a Matthews coefficient of 3.2 Å3 Da(-1) and a solvent content of 61.9%.

Keywords: Arf1; COPI; dimer; p23.

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Figures

Figure 1
Figure 1
Purification of Arf1 and Arf1-GDP. (a) Results of nickel-affinity chromatography. Lane 1, molecular-mass markers (labelled on the left in kDa); lane 2, precipitate of the lysate; lane 3, supernatant of the lysate; lane 4, flowthrough; lanes 5, 6 and 7, elution fractions with 20, 50 and 200 mM imidazole, respectively. The arrow represents Arf1. The amount of Arf1 loaded in lane 7 of the gel was approximately 2.5 µg. (b) Purification of Arf1 by HiLoad 16/60 Superdex 75 pg and SDS–PAGE analysis of the major peak. (c) Purification of Arf1 after nucleotide exchange by HiLoad 16/60 Superdex 75 pg.
Figure 2
Figure 2
(a) Structure of dimeric p23 peptide. (b) Typical crystal of Arf1-GDP in complex with dimeric p23 peptide. (c) Analysis of crystals. Lane M, molecular-mass markers (labelled on the left in kDa); lane 1, Arf1-GDP; lane 2, dimeric p23 peptide; lane 3, crystals.
Figure 3
Figure 3
Self-rotation function for the crystal of Arf1-GDP in complex with dimeric p23 peptide.
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