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Glycoprotein structural genomics. Solving the glycosylation problem

Glycoprotein structural genomics. Solving the glycosylation problem
Glycoprotein structural genomics. Solving the glycosylation problem

Glycoproteins present special problems for structural genomic analysis because they often require glycosylation in order to fold correctly, whereas their chemical and conformational heterogeneity generally inhibits crystallization. We show that the "glycosylation problem" can be solved by expressing glycoproteins transiently in mammalian cells in the presence of the N-glycosylation processing inhibitors, kifunensine or swainsonine. This allows the correct folding of the glycoproteins, but leaves them sensitive to enzymes, such as endoglycosidase H, that reduce the N-glycans to single residues, enhancing crystallization. Since the scalability of transient mammalian expression is now comparable to that of bacterial systems, this approach should relieve one of the major bottlenecks in structural genomic analysis.

0969-2126
267-273
Chang, Veronica T.
e83571e9-6b2f-4f8e-adfc-3fd539e9102e
Crispin, Max
cd980957-0943-4b89-b2b2-710f01f33bc9
Aricescu, A. Radu
8ee3d2bd-7ef7-4e85-862a-e13f48ae0fa6
Harvey, David J.
8bb24417-3852-4b1f-827b-0d5d2c176744
Nettleship, Joanne E.
7055d477-9c3d-46e9-b4a0-2599423afa2f
Fennelly, Janet A.
d3772837-19b5-46fc-9d15-641bf3d11d3e
Yu, Chao
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Boles, Kent S.
5b3a85aa-9b44-44b2-979f-87d20d363396
Evans, Edward J.
d1097f79-7175-4f0b-901c-7c518ed8933e
Stuart, David I.
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Dwek, Raymond A.
d8d9d5f8-f2c0-414e-b6b0-df77a33f0da4
Jones, E. Yvonne
6b9004c9-137b-4f43-8a14-c7c83bcaa4be
Owens, Raymond J.
3aa35960-126a-4ecb-9fb3-088072911a49
Davis, Simon J.
77c9e91c-a2c6-498a-9128-bf164a8da8de
Chang, Veronica T.
e83571e9-6b2f-4f8e-adfc-3fd539e9102e
Crispin, Max
cd980957-0943-4b89-b2b2-710f01f33bc9
Aricescu, A. Radu
8ee3d2bd-7ef7-4e85-862a-e13f48ae0fa6
Harvey, David J.
8bb24417-3852-4b1f-827b-0d5d2c176744
Nettleship, Joanne E.
7055d477-9c3d-46e9-b4a0-2599423afa2f
Fennelly, Janet A.
d3772837-19b5-46fc-9d15-641bf3d11d3e
Yu, Chao
12304bbc-1182-4b35-a3e5-d50719156e2f
Boles, Kent S.
5b3a85aa-9b44-44b2-979f-87d20d363396
Evans, Edward J.
d1097f79-7175-4f0b-901c-7c518ed8933e
Stuart, David I.
2751c230-9d4c-4981-aeb7-cafb51ed749d
Dwek, Raymond A.
d8d9d5f8-f2c0-414e-b6b0-df77a33f0da4
Jones, E. Yvonne
6b9004c9-137b-4f43-8a14-c7c83bcaa4be
Owens, Raymond J.
3aa35960-126a-4ecb-9fb3-088072911a49
Davis, Simon J.
77c9e91c-a2c6-498a-9128-bf164a8da8de

Chang, Veronica T., Crispin, Max, Aricescu, A. Radu, Harvey, David J., Nettleship, Joanne E., Fennelly, Janet A., Yu, Chao, Boles, Kent S., Evans, Edward J., Stuart, David I., Dwek, Raymond A., Jones, E. Yvonne, Owens, Raymond J. and Davis, Simon J. (2007) Glycoprotein structural genomics. Solving the glycosylation problem. Structure, 15 (3), 267-273. (doi:10.1016/j.str.2007.01.011).

Record type: Article

Abstract

Glycoproteins present special problems for structural genomic analysis because they often require glycosylation in order to fold correctly, whereas their chemical and conformational heterogeneity generally inhibits crystallization. We show that the "glycosylation problem" can be solved by expressing glycoproteins transiently in mammalian cells in the presence of the N-glycosylation processing inhibitors, kifunensine or swainsonine. This allows the correct folding of the glycoproteins, but leaves them sensitive to enzymes, such as endoglycosidase H, that reduce the N-glycans to single residues, enhancing crystallization. Since the scalability of transient mammalian expression is now comparable to that of bacterial systems, this approach should relieve one of the major bottlenecks in structural genomic analysis.

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Published date: March 2007

Identifiers

Local EPrints ID: 414581
URI: https://eprints.soton.ac.uk/id/eprint/414581
ISSN: 0969-2126
PURE UUID: 7e0e5aad-5d77-42cb-bdb7-b128af54deee
ORCID for Max Crispin: ORCID iD orcid.org/0000-0002-1072-2694

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Date deposited: 04 Oct 2017 16:30
Last modified: 14 Mar 2019 01:25

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Contributors

Author: Veronica T. Chang
Author: Max Crispin ORCID iD
Author: A. Radu Aricescu
Author: David J. Harvey
Author: Joanne E. Nettleship
Author: Janet A. Fennelly
Author: Chao Yu
Author: Kent S. Boles
Author: Edward J. Evans
Author: David I. Stuart
Author: Raymond A. Dwek
Author: E. Yvonne Jones
Author: Raymond J. Owens
Author: Simon J. Davis

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