کد مقاله | کد نشریه | سال انتشار | مقاله انگلیسی | نسخه تمام متن |
---|---|---|---|---|
8360220 | 1542330 | 2015 | 7 صفحه PDF | دانلود رایگان |
عنوان انگلیسی مقاله ISI
Bacterial expression of the phosphodiester-binding site of the cation-independent mannose 6-phosphate receptor for crystallographic and NMR studies
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کلمات کلیدی
موضوعات مرتبط
علوم زیستی و بیوفناوری
بیوشیمی، ژنتیک و زیست شناسی مولکولی
زیست شیمی
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چکیده انگلیسی
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 â¼60 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 1H-15N HSQC spectra. Additionally, crystallization conditions were identified and a crystallographic data set of the CRD was collected to 1.8Â Ã
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.
ناشر
Database: Elsevier - ScienceDirect (ساینس دایرکت)
Journal: Protein Expression and Purification - Volume 111, July 2015, Pages 91-97
Journal: Protein Expression and Purification - Volume 111, July 2015, Pages 91-97
نویسندگان
Linda J. Olson, Davin R. Jensen, Brian F. Volkman, Jung-Ja P. Kim, Francis C. Peterson, Rebekah L. Gundry, Nancy M. Dahms,