Bacterial expression of the phosphodiester-binding site of the cation-independent mannose 6-phosphate receptor for crystallographic and NMR studies.
Bacterial expression of the phosphodiester-binding site of the cation-independent mannose 6-phosphate receptor for crystallographic and NMR studies.
Protein Expr Purif. 2015 Apr 8;
Authors: Olson LJ, Jensen DR, Volkman BF, Kim JJ, Peterson FC, Gundry RL, Dahms NM
Abstract
The cation-independent mannose 6-phosphate receptor (CI-MPR) is a multifunctional protein that interacts with diverse ligands and plays central roles in autophagy, development, and tumor suppression. By delivering newly synthesized phosphomannosyl-containing acid hydrolases from the Golgi to endosomal compartments, CI-MPR is an essential component in the generation of lysosomes that are critical for the maintenance of cellular homeostasis. The ability of CI-MPR to interact with ~60 different acid hydrolases is facilitated by its large extracellular region, with four out of its 15 domains binding phosphomannosyl residues. Although the glycan specificity of CI-MPR has been elucidated, the molecular basis of carbohydrate binding has not been determined for two out of these four carbohydrate recognition domains (CRD). Here we report expression of CI-MPR's CRD located in domain 5 that preferentially binds phosphodiester-containing glycans. Domain 5 of CI-MPR was expressed in Escherichia coli BL21 (DE3) cells as a fusion protein containing an N-terminal histidine tag and the small ubiquitin-like modifier (SUMO) protein. The His6-SUMO-CRD construct was recovered from inclusion bodies, refolded in buffer to facilitate disulfide bond formation, and subjected to Ni-NTA affinity chromatography and size exclusion chromatography. Surface plasmon resonance analyses demonstrated that the purified protein was active and bound phosphorylated glycans. Characterization by NMR spectroscopy revealed high quality (1)H-(15)N HSQC spectra. Additionally, crystallization conditions were identified and a crystallographic data set of the CRD was collected to 1.8Å resolution. Together, these studies demonstrate the feasibility of producing CI-MPR's CRD suitable for three-dimensional structure determination by NMR spectroscopic and X-ray crystallographic approaches.
PMID: 25863146 [PubMed - as supplied by publisher]
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