Purification of an insulin-related factor secreted by a teratoma-derived mesodermal cell line

Insulin-related factor (IRF), a polypeptide secreted by the mouse teratoma-derived cell line (1246-3A), was purified 3210-fold to homogeneity from 1246-3A conditioned medium using a rapid three-step procedure including cation-exchange chromatography, immunoaffinity chromatography using a monoclonal antibody against porcine insulin coupled to an agarose gel support, and reverse phase high performance liquid chromatography. 10 micrograms of IRF was purified from 6 liters of 1246-3A conditioned medium. Pure IRF appeared as a single band with the same mobility as insulin by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. IRF stimulates cell proliferation of insulin-dependent cell line 1246 and competes with 125I-insulin for binding to 1246 cells; half-maximal growth stimulation and binding competition were achieved at an IRF concentration of 6.5 ng/ml (1.3 nM) and 25 ng/ml (4 nM), respectively, comparable with those for bovine insulin. The biochemical, biological, and immunological characteristics of IRF, as well as its amino acid composition, strongly suggest that it is closely related to pancreatic insulin in structure and function.