Faculty Collaboration Database

Bacterial expression of the phosphodiester-binding site of the cation-independent mannose 6-phosphate receptor for crystallographic and NMR studies. Protein Expr Purif 2015 Jul;111:91-7

Date

04/12/2015

Pubmed ID

25863146

Pubmed Central ID

PMC4417374

DOI

10.1016/j.pep.2015.04.002

Scopus ID

2-s2.0-84927727743 (requires institutional sign-in at Scopus site)   1 Citation

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.

Author List

Olson LJ, Jensen DR, Volkman BF, Kim JJ, Peterson FC, Gundry RL, Dahms NM

Authors

Nancy M. Dahms PhD Professor in the Biochemistry department at Medical College of Wisconsin
Linda J. Olson PhD Assistant Professor in the Pharmacology and Toxicology department at Medical College of Wisconsin
Francis C. Peterson PhD Professor in the Biochemistry department at Medical College of Wisconsin
Brian F. Volkman PhD Professor in the Biochemistry department at Medical College of Wisconsin

MESH terms used to index this publication - Major topics in bold

Binding Sites
Crystallography, X-Ray
Escherichia coli
Gene Expression
Humans
Nuclear Magnetic Resonance, Biomolecular
Receptor, IGF Type 2
Recombinant Proteins