Expression and characterization of recombinant rat placental prolactin-like protein C.
Prolactin-like protein C (PLP-C) is a member of the rat placental family of proteins which are structurally related to pituitary prolactin (PRL). In an effort to characterize the receptor specificity and biological activity of PLP-C, we used a PLP cDNA to express the recombinant protein in a bacterial system. The PLP-C cDNA was modified by oligonucleotide mutagenesis and ligated into a human carbonic anhydrase II (hCAII) expression vector. Following a single step affinity purification, the hCAII-PLP-C fusion protein was digested with enterokinase to release a 25 kDa protein. N-Terminal sequence analysis of the 25 kDa band demonstrated identity with PLP-C. A polyclonal antiserum to the fusion protein cross reacted with seven major proteins in rat placental culture media of which two were the native forms of PLP-C. Recombinant PLP-C was not mitogenic in the Nb2 lymphoma bioassay and did not exhibit high affinity binding to rat PRL receptor. The choice of hCA-II fusion allows for rapid purification of rPLP-C which will aid in further investigation of the biological role of PLP-C.
Conliffe
P R
PR
Department of Pharmacology, University of Florida College of Medicine, Gainesville 32610.
Farmerie
W G
WG
Charles
G D
GD
Buhi
W C
WC
Kelly
P A
PA
Simmen
R C
RC
Shiverick
K T
KT
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015153:Blotting, Western; D002256:Carbonic Anhydrases / Q000235:genetics; D002455:Cell Division / Q000187:drug effects; D018076:DNA, Complementary / Q000737:chemistry / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D015870:Gene Expression; D006801:Humans; D008223:Lymphoma / Q000473:pathology; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010920:Placenta / Q000737:chemistry*; D011257:Pregnancy Proteins / Q000737:chemistry / Q000235:genetics* / Q000494:pharmacology; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000494:pharmacology; D014407:Tumor Cells, Cultured
D018076:DNA, Complementary; D011257:Pregnancy Proteins; C502145:Prl8a3 protein, rat; D011993:Recombinant Fusion Proteins; D002256:Carbonic Anhydrases