Abstract
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.
| Originalsprog | Engelsk |
|---|---|
| Tidsskrift | Preparative Biochemistry and Biotechnology |
| Vol/bind | 21 |
| Udgave nummer | 1 |
| Sider (fra-til) | 35-51 |
| ISSN | 1082-6068 |
| DOI | |
| Status | Udgivet - 1991 |
| Udgivet eksternt | Ja |
Emneord
- analysis Cells,Cultured Chromatography,High Pressure Liquid methods Densitometry Electrophoresis,Polyacrylamide Gel Human interferon Interferon Type I isolation and purification Methods Molecular Structure Neutralization Tests Trophoblast chemistry Record 6 of 7 MEDLINE EXPRESS (R) 1991 1995