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Characterization of recombinantly expressed matrilin VWA domains

Characterization of recombinantly expressed matrilin VWA domains
Characterization of recombinantly expressed matrilin VWA domains
VWA domains are the predominant independent folding units within matrilins and mediate protein–protein interactions. Mutations in the matrilin-3 VWA domain cause various skeletal diseases. The analysis of the pathological mechanisms is hampered by the lack of detailed structural information on matrilin VWA domains. Attempts to resolve their structures were hindered by low solubility and a tendency to aggregation. We therefore took a comprehensive approach to improve the recombinant expression of functional matrilin VWA domains to enable X-ray crystallography and nuclear magnetic resonance (NMR) studies. The focus was on expression in Escherichia coli, as this allows incorporation of isotope-labeled amino acids, and on finding conditions that enhance solubility. Indeed, circular dichroism (CD) and NMR measurements indicated a proper folding of the bacterially expressed domains and, interestingly, expression of zebrafish matrilin VWA domains and addition of N-ethylmaleimide yielded the most stable proteins. However, such proteins did still not crystallize and allowed only partial peak assignment in NMR. Moreover, bacterially expressed matrilin VWA domains differ in their solubility and functional properties from the same domains expressed in eukaryotic cells. Structural studies of matrilin VWA domains will depend on the use of eukaryotic expression systems.
matrilin, extracellular matrix, von willebrand factor a domain
1046-5928
20-28
Becker, Ann-Kathrin A.
a2e4106f-21b5-4cb1-a861-2add4c906fa5
Mikolajek, Halina
c394c255-9248-4217-ace9-4a0382bfc0c5
Werner, Jörn M.
1b02513a-8310-4f4f-adac-dc2a466bd115
Paulsson, Mats
6b6500dc-6e7c-477b-96be-8ebdf82224bf
Wagener, Raimund
401930b6-9833-4d2f-81d0-fc3b1ae020bd
Becker, Ann-Kathrin A.
a2e4106f-21b5-4cb1-a861-2add4c906fa5
Mikolajek, Halina
c394c255-9248-4217-ace9-4a0382bfc0c5
Werner, Jörn M.
1b02513a-8310-4f4f-adac-dc2a466bd115
Paulsson, Mats
6b6500dc-6e7c-477b-96be-8ebdf82224bf
Wagener, Raimund
401930b6-9833-4d2f-81d0-fc3b1ae020bd

Becker, Ann-Kathrin A., Mikolajek, Halina, Werner, Jörn M., Paulsson, Mats and Wagener, Raimund (2015) Characterization of recombinantly expressed matrilin VWA domains. Protein Expression and Purification, 107, 20-28. (doi:10.1016/j.pep.2014.11.005).

Record type: Article

Abstract

VWA domains are the predominant independent folding units within matrilins and mediate protein–protein interactions. Mutations in the matrilin-3 VWA domain cause various skeletal diseases. The analysis of the pathological mechanisms is hampered by the lack of detailed structural information on matrilin VWA domains. Attempts to resolve their structures were hindered by low solubility and a tendency to aggregation. We therefore took a comprehensive approach to improve the recombinant expression of functional matrilin VWA domains to enable X-ray crystallography and nuclear magnetic resonance (NMR) studies. The focus was on expression in Escherichia coli, as this allows incorporation of isotope-labeled amino acids, and on finding conditions that enhance solubility. Indeed, circular dichroism (CD) and NMR measurements indicated a proper folding of the bacterially expressed domains and, interestingly, expression of zebrafish matrilin VWA domains and addition of N-ethylmaleimide yielded the most stable proteins. However, such proteins did still not crystallize and allowed only partial peak assignment in NMR. Moreover, bacterially expressed matrilin VWA domains differ in their solubility and functional properties from the same domains expressed in eukaryotic cells. Structural studies of matrilin VWA domains will depend on the use of eukaryotic expression systems.

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e-pub ahead of print date: 21 November 2014
Published date: March 2015
Additional Information: The research leading to these results has received funding from the European Community’s Seventh Framework Programme under grant agreement n°602300 (SYBIL) and by the Boehringer Ingelheim Fonds. J.M.W. would like to thank the Wellcome Trust for NMR equipment funds at the Southampton NMR center (Grant No. 090658/Z/09/Z) and Stuart Findlow for technical support. We thank the MRC for providing access to the Biomedical NMR Centre at Mill Hill (Grant No. U117533887).
Keywords: matrilin, extracellular matrix, von willebrand factor a domain
Organisations: Molecular and Cellular

Identifiers

Local EPrints ID: 374190
URI: http://eprints.soton.ac.uk/id/eprint/374190
ISSN: 1046-5928
PURE UUID: b9a2e706-0c71-49b4-826d-66aee7cf5916
ORCID for Halina Mikolajek: ORCID iD orcid.org/0000-0003-0776-9974
ORCID for Jörn M. Werner: ORCID iD orcid.org/0000-0002-4712-1833

Catalogue record

Date deposited: 09 Feb 2015 13:52
Last modified: 20 Jul 2019 00:59

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