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The interaction of recombinant decorin with α2HS-Glycoprotein-Implications for structural and functional investigations

Sugars, Rachael V., Waddington, Rachel J. and Embery, Graham 2002. The interaction of recombinant decorin with α2HS-Glycoprotein-Implications for structural and functional investigations. Protein Expression and Purification 25 (1) , pp. 180-188. 10.1006/prep.2002.1625

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Abstract

Isolated protein preparations of the small leucine-rich proteoglycans (SLRPs) associated with mineralized tissues have provided important information in understanding their structural and functional interactions within extracellular matrices and their potential roles in mineralization. Two important SLRPs, decorin and biglycan, copurify following extraction and purification from mineralized tissues using standard procedures, and to overcome this problem decorin was synthesized within a mammalian expression system to obtain pure preparations. The expressed protein was purified from the culture medium using anion-exchange chromatography, and characterization confirmed the presence of a decorin-rich fraction. However, N-terminal sequencing revealed the additional presence of α2HS-glycoprotein (α2HSG), representing approximately 35% of the total purified fraction. The decorin-rich fraction was subjected to selected further purification techniques to separate decorin from α2HSG. Application of the sample at a low concentration (1 mg/ml) to a second anion-exchange procedure and elution over an expanded sodium chloride gradient resulted in a high degree of purity (98%), with a single protein isolate demonstrable by SDS-PAGE. Electroelution achieved partial purification (∼89%), but immunoprecipitation with antibodies against the glycosaminoglycan chain and the polyhistidine tag failed to separate the two proteins. This study suggests there is a strong interaction between recombinantly produced decorin and α2 HSG and highlights the importance of the purification technique to the application of recombinantly produced proteins or those that have been extracted from mineralized tissues for use in structural and functional interactions.

Item Type: Article
Date Type: Publication
Status: Published
Schools: Dentistry
Subjects: R Medicine > RK Dentistry
Uncontrolled Keywords: α2HS glycoprotein; decorin; mineralized tissues; protein expression
Publisher: Elsevier
ISSN: 1046-5928
Last Modified: 04 Jun 2017 04:17
URI: http://orca.cf.ac.uk/id/eprint/36044

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