Heterologous expression and characterization of prolactin-like protein-A. Identification of serum binding proteins.
In this report, we describe the heterologous expression of prolactin-like protein-A (PLP-A) in Chinese hamster ovary (CHO) cells, the characterization of recombinant PLP-A, and the identification of serum PLP-A-binding proteins. CHO cell and native placental PLP-A showed similar immunoreactive characteristics and electrophoretic mobilities. N-terminal sequencing verified the identity and purity of the recombinant PLP-A species and the site of cleavage of the signal peptide from the mature secreted PLP-A species. Recombinant PLP-A lacked activity in standardized prolactin and growth hormone in vitro bioassays. Antibodies generated to recombinant PLP-A facilitated the cellular localization of PLP-A and the identification of high molecular weight PLP-A complexes. Cross-linking analyses of radioiodinated PLP-A with serum harvested from late gestation rats indicated the presence of two major cross-linked complexes migrating under reducing conditions at 130 and 250 kDa and two minor cross-linked complexes migrating at 70 and 110 kDa. Binding of PLP-A to serum proteins was specific for PLP-A and not effectively competed by other members of the prolactin/growth hormone family. The PLP-A binding species were also found in serum from non-pregnant female and male rats.
Deb
S
S
Department of Physiology, University of Kansas Medical Center, Kansas City 66160.
Hamlin
G P
GP
Roby
K F
KF
Kwok
S C
SC
Soares
M J
MJ
D000818:Animals; D015153:Blotting, Western; D016466:CHO Cells; D002352:Carrier Proteins / Q000097:blood* / Q000378:metabolism; D002455:Cell Division / Q000187:drug effects; D006224:Cricetinae; D004247:DNA / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D005822:Genetic Vectors; D008223:Lymphoma; D008297:Male; D008970:Molecular Weight; D010920:Placenta / Q000378:metabolism*; D010957:Plasmids; D011247:Pregnancy; D011257:Pregnancy Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D011388:Prolactin / Q000494:pharmacology; D051381:Rats; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D014162:Transfection; D014407:Tumor Cells, Cultured
D002352:Carrier Proteins; D011257:Pregnancy Proteins; C502143:Prl4a1 protein, rat; D011994:Recombinant Proteins; D011388:Prolactin; D004247:DNA