Purification of 18- and 22-kDa forms of basic fibroblast growth factor from rat cells transformed by the ras oncogene

Abstract

Normal Rat-1 fibroblasts and Rat-1 cells transformed by the H-ras oncogene (Rat-1-EJ) were analyzed for cell-associated growth factor activity. The two cell lines grew at the same rate, but at any given stage of growth the Rat-1-EJ cells synthesized two to four times more cell-associated growth factor activity than did the Rat-1 cells. For each cell line, the level of cell-associated growth factor activity was five to eight times greater at confluent densities compared to sparse densities. Heparin affinity chromatography and Western blot analysis demonstrated that the cell-associated growth factor was basic fibroblast growth factor (bFGF). The bFGF synthesized by the Rat-1-EJ cells appeared in two molecular mass forms, about 40% as an 18-kDa form which comigrated with recombinant bFGF and about 60% as a higher molecular mass doublet of about 22 kDa. The two forms of bFGF were biologically active and could be separated on a Mono S cation exchange column. Separation and purification to homogeneity of both the 18-kDa bFGF and the 22-kDa bFGF doublet were achieved by a combination of CM-Sepharose cation exchange, heparin affinity-fast performance liquid chromatography, and C4 reverse phase high performance liquid chromatography.