| Autor: |
Leibovich, Haim, Raver, Nina, Herman, Asael, Gregoraszczuk, Ewa L., Gootwine, Elisha, Gertler, Arieh |
| Zdroj: |
Protein Expression and Purification; August 2001, Vol. 22 Issue: 3 p489-496, 8p |
| Abstrakt: |
Recombinant bovine Ala-prolactin (PRL) (GenBank Accession No. V00112) in prokaryotic expression plasmid pMON3401 was mutated using a mutagenesis kit, to prepare plasmid encoding ovine PRL (oPRL) (GenBank Accession No. M27057) Escherichia colicells transformed with this latter plasmid overexpressed large amounts of oPRL upon induction with nalidixic acid. The expressed protein, found in inclusion bodies, was refolded and purified to homogeneity on a Q-Sepharose column, yielding an electrophoretically pure fraction composed of over 98% monomeric protein of the expected molecular mass of ∼23 kDa. The biological activity of the recombinant oPRL after proper renaturation was evidenced in vitroby its ability to stimulate proliferation of rat lymphoma Nb2cells possessing PRL receptors, to stimulate luciferase activity in HEK 293 cells transiently transfected with oPRL receptors, and to induce progesterone secretion in primary cultures of luteal cells obtained from midpregnant ewes. In contrast to ovine growth hormone or ovine placental lactogen, recombinant oPRL had no galactopoietic effect in lactating ewes. |
| Databáze: |
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