Recombinant Feline Oviductin – A Powerful Tool for Functional IVF Studies in the Domestic Cat

Authors


Author's address (for correspondence): Alexandra Hachen, Leibniz Institute for Zoo and Wildlife Research, PF 700430, 10324 Berlin, Germany. E-mail: hachen@izw-berlin.de

Contents

Oviductins are highly conserved proteins that seem to play an important role during fertilization and embryo development. A universal characteristic of oviductin is its association with the zona pellucida of ovulated oocytes, and several functional studies indicate an influence of this glycoprotein on reproductive events in mammals. The objective of this study was to produce recombinant feline oviductin in prokaryotic as well as eukaryotic cells as a prerequisite for analysing its influence on IVF in the domestic cat. Oviductin sequence was cloned into pET21b vector and transformed to E. coli to produce a recombinant His-tagged, non-glycosylated protein. Oviductin was isolated from E. coli lysate using anion exchange chromatography followed by immobilized metal ion affinity chromatography (IMAC). Western blot analysis of affinity purified fractions resulted in one clear band corresponding to the expected size of ~67 kDa. The corresponding band of a coomassie-stained gel was analysed by mass spectrometry. Oviductin was identified with over 50 peptides covering 72% of the whole protein sequence. To obtain a glycosylated form of oviductin, eukaryotic cells were stable transfected with pSecTag/HygroA vector containing the oviductin gene sequence. The recombinant His-tagged protein was harvested from a serum- and protein-free cell culture medium. Mass spectrometry analyses of protein bands obtained after separation of the medium by SDS-PAGE detected oviductin peptides in protein bands of ~70, ~85 and ~170 kDa. With prokaryotic as well as eukaryotic produced recombinant feline oviductin, we are now able to use the protein for further functional IVF studies in the domestic cat.

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