Isolation, purification and biochemical characterization of human placental interferons by tandem high-performance affinity chromatography.
Human placental trophoblasts, fibroblasts and the trophoblast-derived malignant cell JAR are potent producers of interferons (IFNs) when stimulated with Sendai virus. The three cell lines produced different levels and compositions of IFN-alpha subtypes and IFN-beta. Anti-IFN globulins, Cibacron Blue F3GA and Concanavalin A were covalently immobilized on pressure-stable, macroporous polymeric matrices derivatized with vinyl sulphone (HEMA-BIO 1000 VS and HEMA 1000 VS). These supports were packed in biocompatible PEEK columns and were coupled with switching valves, to develop a tandem high-performance affinity chromatographic (HPAC) method for the isolation, purification and biochemical characterization of the IFNs produced in Sendai virus-stimulated human placental trophoblasts, fibroblasts and trophoblast-derived malignant cell, JAR, cultures. Silver-stained SDS-PAGE and gel densitometric analysis revealed the purity of the purified proteins to be between 94 and 98%. Specific activities of the purified IFNs ranged between 0.37-2.76 x 10(8) IU/mg of protein with cumulative recoveries between 90 and 92.2%. The purified IFN components exhibited quantitatively different antiviral activities in human and bovine cell lines. The utility of the tandem method for the purification and characterization of human type 1 IFNs produced from other cell lines are also discussed.
Aboagye-Mathiesen
G
G
Department of Virus and Cancer, Danish Cancer Society, Aarhus.
Tóth
F D
FD
Dalsgaard
A M
AM
Petersen
P M
PM
Zachar
V
V
Ebbesen
P
P
D002846:Chromatography, Affinity; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D005347:Fibroblasts / Q000737:chemistry; D006801:Humans; D007372:Interferons / Q000302:isolation & purification*; D010920:Placenta / Q000737:chemistry*; D011247:Pregnancy; D014327:Trophoblasts / Q000737:chemistry
D007372:Interferons