Decidual prolactin-related protein: heterologous expression and characterization.
As a first step in understanding the role of decidual PRL-related protein (dPRP) during pregnancy, we have generated recombinant dPRP protein. In this report, we present data on the generation, purification, and characterization of recombinant dPRP protein. The dPRP complementary DNA was subcloned into the pMSXND vector, and the vector was transfected into Chinese hamster ovary (CHO) cells by electroporation. After appropriate selection, amplification, and induction procedures, recombinant dPRP was purified from conditioned medium of the CHO-dPRP cells using ultrafiltration, size-exclusion chromatography, and reverse phase HPLC. Recombinant dPRP was found to possess electrophoretic mobility, immunoreactivity, and N-terminal amino acid sequence identical to those of dPRP isolated from decidual tissue. Polyclonal antibodies were generated to the recombinant dPRP and used for Western blot analysis. dPRP is capable of binding heparin, and a significant fraction of synthesized dPRP resides within the decidual extracellular matrix. Recombinant dPRP failed to bind to PRL receptors and showed no stimulatory activity in the PRL-dependent rat Nb2 lymphoma cell proliferation assay. Additional studies have shown that heterologous expression of dPRP in CHO cells significantly increased the ability of CHO cells to form tumors in athymic mice. In conclusion, recombinant dPRP possesses characteristics similar to those of dPRP of decidual origin and is a heparin-binding protein that may facilitate the establishment of pregnancy.
Rasmussen
C A
CA
Department of Physiology, University of Kansas Medical Center, Kansas City 66160, USA.
Hashizume
K
K
Orwig
K E
KE
Xu
L
L
Soares
M J
MJ
D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D004347:Drug Interactions; D006493:Heparin / Q000494:pharmacology; D051379:Mice; D008819:Mice, Nude; D009389:Neovascularization, Pathologic / Q000473:pathology; D018919:Neovascularization, Physiologic / Q000187:drug effects; D011388:Prolactin / Q000031:analogs & derivatives* / Q000302:isolation & purification / Q000378:metabolism / Q000494:pharmacology; D051381:Rats; D011919:Rats, Inbred Strains; D011994:Recombinant Proteins; D012756:Sheep
C489796:Dtprp protein, mouse; C079559:Prl8a2 protein, rat; D011994:Recombinant Proteins; D011388:Prolactin; D006493:Heparin