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 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.

Figure 1

Electron micrographs of elastic fibers. Left: Standard transmission microscopy of developing lung showing an elastic fiber consisting of elastin (E) and microfibrils (MF). The microfibrils remain on the periphery of the fiber and are thought to help structure the fiber and assist in elastin crosslinking. Right: A similar elastic fiber visualized using quick-freeze, deep etch microscopy [78]. Unlike standard transmission microscopy, quick-freeze, deep etch images provide insight into organization of elastin within the fiber. The plasma membrane of an adjacent cell runs from top left to lower right and is indicated by PM.

Figure 2

Schematic diagram of exon and domain structure of human tropoelastin. Colored squares represent lysine-crosslinking domains that contain prolines (KP) or are enriched in alanines (KA). White squares are hydrophobic sequences. The unique C-terminal region important for fiber assembly is in blue. Stars indicate exons that are alternatively spliced. Human elastin lacks exons 34 and 35 found in other mammalian elastins.

Figure 3

Modification of lysine side chains in crosslinking of elastin. Crosslinking of elastin monomers is initiated by the oxidative deamination of lysine side chains by the enzyme lysyl oxidase in a reaction that consumes molecular oxygen and releases ammonia. The aldehyde that is formed can condense with another modified side chain aldehyde (1) to form the bivalent aldol condensation product (ACP) crosslink. Reaction with the amine of an unmodified side chain through a Shiff base reaction (2) produces dehydrolysinonorleucine (dLNL). ACP and dLNL can then condense to form the tetrafunctional crosslink desmosine or isodesmosine.

Figure 4

Amino acid composition of elastin. Amino acid composition of human, bovine, and mouse elastins expressed as residues per molecule (Number) and residues per 1000 residues (res/1000). Composition was determined from sequences found in Genebank. Lysine values are estimates based on full crosslinking.

Figure 5

Temporal expression profile of elastin in mouse aorta. Expression of elastin as determined by oligonucleotide microarray (median normalized values) in developing mouse aorta. The pattern shows a major increase in expression beginning around embryonic day 14 and continuing through postnatal days 7–10. Thereafter, expression rapidly decreases to low levels that persist into the adult period.

Figure 6

Effects of cell density on elastin synthesis. Tropoelastin production was monitored in cultures of ligamentum nuchae fibroblasts as cells were grown from sparse density (3 × 10⁵ cells/100 dish) to confluence (~7 × 10⁶ cells per dish). Tropoelastin (soluble elastin--squares) was not detected until cells approached confluence, indicated by a reduction in the rate of increase in cell number (circles) and a diminution in thymidine incorporation (triangles). Data from [65], used with permission.

Figure 7

Binding of tropoelastin in cell culture medium to microtiter plates. Shown is a typical standard curve generated with purified tropoelastin dissolved in DMEM-10% calf serum and developed with tropoelastin antibody. Serial dilution of culture medium from fetal bovine chondrocytes is shown in the insert. From [45]. Used with permission.