PUBLICATION
Characterization of recombinantly expressed matrilin VWA domains
- Authors
- Becker, A.K., Mikolajek, H., Werner, J.M., Paulsson, M., Wagener, R.
- ID
- ZDB-PUB-141203-10
- Date
- 2015
- Source
- Protein Expression and Purification 107: 20-8 (Journal)
- Registered Authors
- Keywords
- Extracellular matrix, Matrilin, Von Willebrand factor A domain
- MeSH Terms
-
- Animals
- Circular Dichroism
- Crystallography, X-Ray
- Escherichia coli/genetics
- Escherichia coli/metabolism
- Humans
- Matrilin Proteins/chemistry*
- Matrilin Proteins/genetics*
- Matrilin Proteins/isolation & purification
- Matrilin Proteins/metabolism
- Protein Conformation
- Protein Structure, Tertiary
- Zebrafish
- PubMed
- 25462806 Full text @ Protein Expr. Purif.
Citation
Becker, A.K., Mikolajek, H., Werner, J.M., Paulsson, M., Wagener, R. (2015) Characterization of recombinantly expressed matrilin VWA domains. Protein Expression and Purification. 107:20-8.
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 E. 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.
Genes / Markers
Expression
Phenotype
Mutations / Transgenics
Human Disease / Model
Sequence Targeting Reagents
Fish
Orthology
Engineered Foreign Genes
Mapping