63] Purification of human fibroblast interferon by high-performance liquid chromatography

Abstract

This chapter presents a procedure combining the techniques of affinity chromatography and high-performance liquid chromatography (HPLC) for the preparation of fibroblast interferon from serum-containing medium. The purified fibroblast interferon is homogeneous by several criteria of molecular characterization and has a specific activity of approximately 4 x l0 8 units/mg. The purified protein is present in a completely volatile, detergent-free solvent. From several possible approaches, the combination of Blue Dextran-Sepharose affinity chromatography with reverse-phase HPLC at acidic pH is outlined. In contrast to leukocyte interferon, it is not possible to use neutral pH conditions for the HPLC steps because fibroblast interferon rapidly lost activity when organic solvents (propanols, acetone, Methylcellosolve, and such) are present. The data for the homogeneous protein is a single band on sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis, which coincided with the recoverable antiviral activity, and a single protein peak in the last chromatographic step, which coincided with the antiviral activity. The purified preparations are characterized by amino acid analyses, amino sugar analyses, end-group determinations, tryptic maps, and amino acid sequencing.