Purification of human placental trophoblast interferon by two-dimensional high performance liquid chromatography

George Aboagye-Mathiesen, Ferenc D. Toth, Claus Juhl, Niels Nørskov-Lauritsen, Peter M. Petersen, Peter Ebbesen

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Abstract

Human placental trophoblast challenged with Sendai virus induced IFNs mainly of the β-type (75%) and relatively low levels of the α-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-β) 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-β 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-β had a specific activity of 1.03 x 108 lU/mg of protein and the overall recovery was 81% of the total IFN-β activity in the crude preparation and 61% of the total IFN activity.

Original languageEnglish
Pages (from-to)35-51
Number of pages17
JournalPreparative Biochemistry
Volume21
Issue number1
DOIs
Publication statusPublished - Mar 1991

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ASJC Scopus subject areas

  • Biochemistry
  • Genetics

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