Methods in elastic tissue biology: Elastin isolation and purification

Abstract

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 call 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.

Oxidative and Nitrosative Modifications of Tropoelastin Prevent Elastic Fiber Assembly in Vitro

“…Other components, such as glycosaminoglycans (GAGs), lysyl oxidase (LOX), and other elastin-binding proteins are also present in elastic fibers (1)(2)(3)(4).…”

Age Estimation by Using Aspartic Acid Racemization from Purified Elastin of Aorta

“…Elastin can be easily used for purification which is used to heat and alter condition of pH i.e. hot alkali being considered for purified elastin tissue (Mecham 2008). There has been a comparative method of purification to study differences in separation and emzyme digestion to take away non-elastin contaminants (Daamen et al 2001).…”

Investigation of Long-Term Retention of Unchanged (−)-N-{2-[(R)-3-(6,7-Dimethoxy-1,2,3,4-tetrahydroisoquinoline-2-carbonyl)piperidino]ethyl}-4-fluorobenzamide, A Novel “Funny” If Current Channel Inhibitor, and Its Metabolites in the Eyeball and Thoracic Aorta of Rats

“…6), and it might represent the binding between YM758-based structures and amino acid residues derived from elastin fiber. For clarifying the binding mechanism, the elastin components were purified under the alkaline condition by using thoracic aorta excised from rats after dosing of YM758, and they were sequentially hydrolyzed under the acidic condition (Mecham RP, 2008). The hydrolyzed elastin was subject to HPLC-MS analyses after forming the corresponding amino acid derivatives, and finally the peak corresponding to the molecular weight of 472 was detected (data not shown).…”