Isolation, purification and characterization of the intermediate filament protein desmin from porcine smooth muscle.

Desmin was isolated from porcine stomach smooth muscle, which had been treated with 0.6 M KCl in the presence of Triton X-100, by extraction with 6 M urea and chromatographed on CM-Sepharose CL-6B at pH 5. After delipidation with chloroform-methanol, the protein was purified by affinity chromatography on arginine methylester Sepharose 4B and single-stranded DNA-cellulose, respectively. Chromatography on single-stranded DNA-cellulose removed a considerable amount of vimentin which had been extracted and enriched together with desmin. The molecular weight of the purified desmin was 55,000 as determined by polyacrylamide gradient slab gel electrophoresis in the presence of Na-dodecylsulfate. Two-dimensional polyacrylamide gel electrophoresis revealed one major polypeptide of pI 5.3 to 5.4 accompanied by two to three acidic, isoelectric variants. During incubation in the presence of 150 mM KCl, desmin assembled into 10 nm filaments. This method allows the isolation of large amounts of pure desmin in a relatively short time with only minimal denaturation of the protein.