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 SLRP's, 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 alpha(2)HS-glycoprotein (alpha(2)HSG), representing approximately 35% of the total purified fraction. The decorin-rich fraction was subjected to selected further purification techniques to separate decorin from alpha(2)HSG. 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 (similar to89%), 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 alpha(2)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. (C) 2002 Elsevier Science (USA).