Rapid purification of leukocyte interferons by high-performance liquid chromatography

Abstract

Many species of interferon have been identified and purified from human leukocytes, and genes for these interferons have been cloned in bacterial systems. However, it is still unclear as to how the expression of each of the different leukocyte interferons is regulated and how they function in the natural system. In preparation for a study of the in vivo role of individual leukocyte interferons we have developed a rapid and convenient high-performance liquid chromatographic method for purifying several species of human leukocyte interferon. Interferon produced by the induction of leukocytes (obtained from buffy coats of normal donors) with Sendai virus and partially purified by ethanol fractionation was further purified by size-exclusion high-performance liquid chromatography (on two Waters,I-125 columns) in 0.05 M sodium phosphate buffer, pH 6, containing 0.2 M sodium chloride. The flow-rate was 2 ml/min and interferon activity (antiviral) was eluted from the columns in less than 15 min in a single fraction well resolved from high-molecular-weight contaminants. This material was then purified by reversed-phase high-performance liquid chromatography on a wide-pore diphenyl column (Whatman Protesil 300) with a gradient system at pH 2.4, consisting of (A) 0.05 M KH2PO4—methoxyethanol (19:1) and (B) 1-propanol—methoxyethanol (19:1). Several peaks of interferon activity were distinguishable in this system. Analysis of the pooled peaks of interferon activity by SDS polyacrylamide gel electrophoresis confirmed the presence of more than one form of leukocyte interferon. The specific activity of the purified leukocyte interferers was ca. 108 units/mg protein.