Large-scale preparation of recombinant ovine prolactin and determination of its in vitro and in vivo activity

Recombinant bovine Ala-prolactin (PRL) (GenBank Accession No. V00112) in pr okaryotic expression plasmid pMON3401 was mutated using a mutagenesis kit, to prepare plasmid encoding ovine PRL (oPRL) (GenBank Accession No. M27057) Escherichia coli cells transformed with this latter plasmid overexpressed large amounts of oPRL upon induction with nalidixic acid. The expressed pro tein, found in inclusion bodies, was refolded and purified to homogeneity o n a Q-Sepharose column, yielding an electrophoretically pure fraction compo sed of over 98% monomeric protein of the expected molecular mass of similar to 23 kDa. The biological activity of the recombinant oPRL after proper re naturation was evidenced in vitro by its ability to stimulate proliferation of rat lymphoma Nb-2 cells possessing PRL receptors, to stimulate lucifera se activity in HEK 293 cells transiently transfected with oPRL receptors, a nd to induce progesterone secretion in primary cultures of luteal cells obt ained from midpregnant ewes. In contrast to ovine growth hormone or ovine p lacental lactogen, recombinant oPRL had no galactopoietic effect in lactati ng ewes. (C) 2001 Academic Press.