Purification of human placental trophoblast interferon by two-dimensional high performance liquid chromatography.
Human placental trophoblast challenged with Sendai virus induced IFNs mainly of the beta-type (75%) and relatively low levels of the alpha-type (25%). A two-step high performance liquid chromatographic procedure ("two-dimensional HPLC") has been developed for the complete purification of the placental trophoblast interferon beta (tro-IFN-beta) from serum-containing culture supernatant. The method involved a combination of high performance liquid affinity chromatography (HPLAC) on Cibacron Blue 3GA immobilized on an activated pressure stable macroporous synthetic polymer, 2-hydroxyethyl methacrylate vinyl sulphone (HEMA-BIO 1000 VS), as the first dimension and reversed-phase high performance liquid chromatography (RP-HPLC) on Separon SGX C-18 as the second. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblot experiments showed that the tro-IFN-beta was present as a 24 kDa protein. Densitometric scanning analysis of Coomassie-stained gel revealed the purity of the final preparation to be greater than 99%. The purified tro-IFN-beta had a specific activity of 1.03 x 10(8) IU/mg of protein and the overall recovery was 81% of the total IFN-beta activity in the crude preparation and 61% of the total IFN activity.
Aboagye-Mathiesen
G
G
Danish Cancer Society, Department of Virus and Cancer, Aarhus.
Toth
F D
FD
Juhl
C
C
Nørskov-Lauritsen
N
N
Petersen
P M
PM
Ebbesen
P
P
D002478:Cells, Cultured; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D003720:Densitometry; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D007370:Interferon Type I / Q000302:isolation & purification*; D015394:Molecular Structure; D009500:Neutralization Tests; D014327:Trophoblasts / Q000737:chemistry*
D007370:Interferon Type I