Purification of the intermediate filament protein vimentin from Ehrlich ascites tumor cells.

Two procedures have been used for the purification of the intermediate filament protein vimentin from Ehrlich ascites tumor (EAT) cells grown in vitro. In one procedure, vimentin was first incorporated into residual cell structures by extracting EAT cells with Triton X-100 in the presence of M g ’ and then solubilized with low ionic strength buffer in the absence of M g + . In the second procedure, which led to the purification of both vimentin and the Ca2+-activated proteinase specific for this intermediate filament protein, both components were immediately solubilized by extracting EAT cells with hypotonic buffer in the absence of Triton X-100. In both procedures, a vimentin-enriched fraction was obtained by precipitating vimentin with (N&)2S04 at 23% saturation. Following lyophilization and delipidation with chloroform/methanol, vimentin was purified by column chromatography on DEAE-Sepharose C16B in 6 M urea. The first procedure resulted in a 33-fold purification of vimentin in a yield of 39%. The same method was also used to prepare [3H]vimentin from EAT cells labeled with 3H-amino acids and resulted in a specific activity of 8 X lo6 cpm/mg purified vimentin. By two-dimensional polyacrylamide gel electrophoresis only one protein species was seen following staining with silver. The purified protein had a subunit molecular weight of 58,000 and consisted of several isoelectric variants (PI 5.3). In the absence of urea and at Iow saIt concentration the purified vimentin was highly aggregated and had a sedimentation coefficient of 8 s.