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. 2018 May 1;74(Pt 5):322-326.
doi: 10.1107/S2053230X18005551. Epub 2018 Apr 25.

The CD163 long-range scavenger receptor cysteine-rich repeat: expression, purification and X-ray crystallographic characterization

Rui Li  1 , Hongfang Ma  1 , Longguang Jiang  2 , Songlin Qiao  1 , Yubao Zhi  1 , Mingdong Huang  2 , Ruiguang Deng  1 , Gaiping Zhang  1
Affiliations

The CD163 long-range scavenger receptor cysteine-rich repeat: expression, purification and X-ray crystallographic characterization

Rui Li et al. Acta Crystallogr F Struct Biol Commun. .

Abstract

Scavenger receptors (SRs) play critical roles in various physiological and pathological pathways. One of them, CD163, is a multifunctional endocytic receptor and is characterized by a long-range scavenger receptor cysteine-rich (SRCR) repeat. However, the structural and functional details of this long-range SRCR repeat have not yet been elucidated. In this study, the CD163 long-range SRCR repeat was expressed in Drosophila Schneider 2 cells. The recombinant protein was homogeneous after purification by metal-affinity, cation-exchange and size-exclusion chromatography. Single crystals were obtained using 20% PEG 4000, 0.15 M potassium sodium tartrate tetrahydrate pH 8.5 and diffracted to 3.30 Å resolution. As the first view of a long-range SRCR repeat, this work lays the structural basis for a deep understanding of SRs and their multiple functions.

Keywords: CD163; SRCR repeat; X-ray crystallography; long-range scavenger receptor cysteine-rich repeat; scavenger receptor.

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Figures

Figure 1
Figure 1
Production of recombinant CD163 long-range SRCR repeat. (a) Schematic diagram of CD163 and its long-range SRCR repeat. CD163 consists of nine SRCR domains (1–9), two proline–serine–threonine (PST)-rich motifs (PST I and II), a transmembrane domain (TM) and a cytoplasmic tail. The CD163 long-range SRCR repeat spans from SRCR5 to SRCR9. (b) Size-exclusion chromatography of the CD163 long-range SRCR repeat on a calibrated Superdex 200 10/300 GL column. The target protein eluted at a retention volume of 14 ml, corresponding to approximately 67 kDa. (c) Characterization of recombinant CD163 long-range SRCR repeat by 12% SDS–PAGE after three-step purification. Lane 1, the CD163 long-range SRCR repeat under nonreducing conditions. Lane M, molecular-mass marker (labelled in kDa). Lane 2, the CD163 long-range SRCR repeat under reducing conditions. Lane 3, PNGase F-treated CD163 long-range SRCR repeat. PNGase F (about 36 kDa) is not obvious in the figure.
Figure 2
Figure 2
Crystallization of recombinant CD163 long-range SRCR repeat. (a) Crystals of the CD163 long-range SRCR repeat (circled in red). (b) SDS–PAGE analysis of the washed crystals. Lane M, molecular-mass marker (labelled in kDa). Lane 1, crystals of the CD163 long-range SRCR repeat dissolved in water.
Figure 3
Figure 3
Representative diffraction image obtained from crystals of the CD163 long-range SRCR repeat. The inset shows a magnification of the diffraction pattern in the red box.
See this image and copyright information in PMC

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