Universität zu Köln

Becker, Ann-Kathrin A., Mikolajek, Halina, Werner, Joern M., Paulsson, Mats and Wagener, Raimund (2015). Characterization of recombinantly expressed matrilin VWA domains. Protein Expr. Purif., 107. S. 20 - 29. SAN DIEGO: ACADEMIC PRESS INC ELSEVIER SCIENCE. ISSN 1096-0279

Full text not available from this repository.

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. (C) 2014 Published by Elsevier Inc.

Item Type:	Journal Article
Creators:	Creators Email ORCID ORCID Put Code Becker, Ann-Kathrin A. UNSPECIFIED UNSPECIFIED UNSPECIFIED Mikolajek, Halina UNSPECIFIED UNSPECIFIED UNSPECIFIED Werner, Joern M. UNSPECIFIED UNSPECIFIED UNSPECIFIED Paulsson, Mats UNSPECIFIED UNSPECIFIED UNSPECIFIED Wagener, Raimund UNSPECIFIED UNSPECIFIED UNSPECIFIED
URN:	urn:nbn:de:hbz:38-411984
DOI:	10.1016/j.pep.2014.11.005
Journal or Publication Title:	Protein Expr. Purif.
Volume:	107
Page Range:	S. 20 - 29
Date:	2015
Publisher:	ACADEMIC PRESS INC ELSEVIER SCIENCE
Place of Publication:	SAN DIEGO
ISSN:	1096-0279
Language:	English
Faculty:	Unspecified
Divisions:	Unspecified
Subjects:	no entry
Uncontrolled Keywords:	Keywords Language MULTIPLE EPIPHYSEAL DYSPLASIA; A-DOMAIN; HAND OSTEOARTHRITIS; PROTEIN SOLUBILITY; COLLAGEN FIBRILS; CARTILAGE; MUTATION; REGION; CHONDRODYSPLASIAS; STABILITY Multiple languages Biochemical Research Methods; Biochemistry & Molecular Biology; Biotechnology & Applied Microbiology Multiple languages
URI:	http://kups.ub.uni-koeln.de/id/eprint/41198