The extracellular domain of rabbit prolactin receptor (rbPRLR-ECD) expressed in an insect/baculovirus expression system was purified by affinity chromatography on immobilized PRL followed by gel filtration. The purified protein was over 90% homogeneous as indicated by SDS-PAGE in the presence or absence of reducing agent, and by chromatography on a Superdex column. Its molecular mass determined by SDS-PAGE was 32 kDa, and by gel filtration, 27 kDa. Both values are higher than the 22.8 kDa deduced from the cDNA sequence, indicating extensive glycosylation. The K a value for interaction with ovine (o) PRL was 25.4 nM−1, but even at high rbPRLR-ECD:hormone molar ratios, the stoichiometry of interaction with oPRL or human growth hormone indicated formation of only 1:1 complexes, in contrast to human growth hormone (hGH)-ECD which forms 2:1 complexes with hGH.
