Purification of an acid-stable bovine leukocyte interferon.
Bovine leukocyte interferon (BoL-IFN), produced in bovine peripheral blood leukocytes after priming and induction with Sendai virus, was concentrated by precipitation with KSCN (pH 3.5) and purified by gel column chromatography. Recovery of BoL-IFN from precipitation was higher when crude BoL-IFN containing more fetal bovine serum (FBS) was used. However, purity of BoL-IFN recovered from the gel filtration column was highest when crude BoL-IFN with no FBS was used. The use of 25% ethylene glycol in the column elution buffer resulted in over 93% recovery of the applied IFN activity, versus only 25% when buffer contained no ethylene glycol. Column-purified BoL-IFN was further concentrated by ultrafiltration and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) in denaturing buffer. When crude BoL-IFN containing no FBS was used for purification, BoL-IFN from a selected column fraction applied to SDS-PAGE resulted in a single narrow band with an apparent molecular weight (MW) of 19,000 Da. Extraction of the SDS-PAGE gel resulted in a single peak of IFN activity indicating identity of the activity and the polypeptide. This proved to be a practical method for obtaining sufficient quantities of purified natural BoL-IFN for use in the production of monoclonal antibodies to BoL-IFN and other biological experiments.
Abolhassani
M
M
Department of Large Animal Medicine, College of Veterinary Medicine, University of Georgia, Athens 30602.
Jacobsen
K L
KL
D000818:Animals; D002417:Cattle / Q000097:blood*; D002850:Chromatography, Gel; D004591:Electrophoresis, Polyacrylamide Gel; D007369:Interferon Inducers; D016898:Interferon-alpha / Q000737:chemistry / Q000302:isolation & purification*; D007962:Leukocytes / Q000378:metabolism*; D008970:Molecular Weight
D007369:Interferon Inducers; D016898:Interferon-alpha