Methods in elastic tissue biology: elastin isolation and purification.
Elastin provides recoil to tissues subjected to repeated stretch, such as blood vessels and the lung. It is encoded by a single gene in mammals and is secreted as a 60-70 kDa monomer called tropoelastin. The functional form of the protein is that of a large, highly crosslinked polymer that organizes as sheets or fibers in the extracellular matrix. Purification of mature, crosslinked elastin is problematic because its insolubility precludes its isolation using standard wet-chemistry techniques. Instead, relatively harsh experimental approaches designed to remove non-elastin 'contaminates' are employed to generate an insoluble product that has the amino acid composition expected of elastin. Although soluble, tropoelastin also presents problems for isolation and purification. The protein's extreme stickiness and susceptibility to proteolysis requires careful attention during purification and in tropoelastin-based assays. This article describes the most common approaches for purification of insoluble elastin and tropoelastin. It also addresses key aspects of studying tropoelastin production in cultured cells, where elastin expression is highly dependent upon cell type, culture conditions, and passage number.
Mecham
Robert P
RP
Department of Cell Biology and Physiology, Washington University School of Medicine, 660 South Euclid Avenue, St. Louis, MO 63110, USA. bmecham@wustl.edu
D000596:Amino Acids / Q000737:chemistry; D000818:Animals; D002417:Cattle; D002478:Cells, Cultured; D003895:Desmosine / Q000032:analysis / Q000737:chemistry; D004549:Elastin / Q000737:chemistry* / Q000302:isolation & purification* / Q000648:ultrastructure; D006801:Humans; D051379:Mice; D012995:Solubility
D000596:Amino Acids; D003895:Desmosine; D004549:Elastin