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Cloning, Expression, and Purification of the Glycosylated Transmembrane Protein, Cation-Dependent Mannose 6-Phosphate Receptor, from Sf9 Cells Using the Baculovirus System

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The Surfaceome

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1722))

Abstract

The cation-dependent mannose 6-phosphate receptor (CD-MPR) is a single-pass type I membrane protein. This protein functions to transport lysosomal enzymes displaying phosphomannosyl residues from the Golgi complex and the cell surface to the lysosome. This glycosylated protein contains three disulfide bridges in its 159-residue extracytoplasmic domain. One of the problems with studying eukaryotic membrane proteins is isolating sufficient quantities. Structural studies typically require several hundred milligrams of highly purified protein. Here we present a method to isolate milligram quantities of CD-MPR/Asn81 suitable for structural studies.

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Acknowledgment

This work supported by NIH grant R01 DK042667 to NMD.

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Authors and Affiliations

  1. Department of Biochemistry, Medical College of Wisconsin, Milwaukee, WI, USA

    Linda J. Olson & Nancy M. Dahms

Authors
  1. Linda J. Olson
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  2. Nancy M. Dahms
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Corresponding author

Correspondence to Linda J. Olson .

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Editors and Affiliations

  1. School of Biomedical Sciences, LKS Faculty of Medicine, The University of Hong Kong, Hong Kong, SAR, China

    Kenneth R. Boheler

  2. Department of Biochemistry, Medical College of Wisconsin, Milwaukee, Wisconsin, USA

    Rebekah L. Gundry

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Olson, L.J., Dahms, N.M. (2018). Cloning, Expression, and Purification of the Glycosylated Transmembrane Protein, Cation-Dependent Mannose 6-Phosphate Receptor, from Sf9 Cells Using the Baculovirus System. In: Boheler, K., Gundry, R. (eds) The Surfaceome. Methods in Molecular Biology, vol 1722. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7553-2_7

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  • DOI: https://doi.org/10.1007/978-1-4939-7553-2_7

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  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-7551-8

  • Online ISBN: 978-1-4939-7553-2

  • eBook Packages: Springer Protocols

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