64] Purification of human fibroblast interferon produced in the absence of serum by cibacron blue F3GA-agarose and high-performance liquid chromatography

Abstract

This chapter describes the procedure that combines the use of affinity chromatography and high-performance liquid chromatography to purify human fibroblast interferon produced in the absence of serum. The final product has a specific activity of about 3 x 10 8 units per milligram of protein and is homogeneous by the criteria of sodium dodecyl sulfate (SDS)-gel electrophoresis, amino acid analysis, and amino acid sequencing. In addition, this procedure has the advantage that the pure interferon is left in a volatile buffer, and thus is easily concentrated or dried for further studies. Affinity chromatography provides a high purification factor, but yields a dilute solution of interferon in 50% ethylene glycol. The final product is then obtained in concentrated form by HPLC. The interferon may be recovered salt- and solvent- free by evaporation because only volatile eluents are used. As proof of homogeneity, a single band is obtained on SDS-polyacrylamide gel electrophoresis as well as a single NH2-terminal amino acid sequence.