Production of recombinant goldfish prolactin and its applications in radioreceptor binding assay and radioimmunoassay.
Goldfish prolactin cDNA was subcloned into a pRSET A vector and expressed in Escherichia coli. Recombinant goldfish prolactin was expressed mainly as insoluble inclusion bodies in the form of N-terminal 6x His-tagged fusion protein. This fusion protein was purified, refolded, and (125)I-labeled to generate a radioligand for receptor binding and validation of a radioimmunoassay for goldfish prolactin. Using goldfish gill membrane as the substrate for prolactin receptor binding, both recombinant and native forms of goldfish prolactin were effective in displacing the specific binding of the radioligand in a similar dose range, suggesting that the fusion protein was refolded properly and could be recognized by goldfish prolactin receptors. To quantify prolactin contents in biological samples from the goldfish, a radioimmunoassay using the (125)I-labeled recombinant prolactin as a tracer was established. This assay was shown to be selective for goldfish prolactin without cross-reactivity with mammalian prolactin and pituitary hormones from other fish species (e.g., growth hormone and gonadotropin II). This newly validated assay system was used to investigate neuroendocrine and signal transduction mechanisms regulating prolactin release in the goldfish. In this case, the Ca(2+) ionophore A23187 and protein kinase C activator TPA were effective in elevating basal levels of prolactin secretion in perifused goldfish pituitary cells. In parallel studies using a static incubation approach, somatostatin and dopamine, but not vasoactive intestinal polypeptide, were inhibitory to basal prolactin release in goldfish pituitary cells. These results suggest that somatostatin and dopamine may serve as negative regulators of basal prolactin secretion and that extracellular Ca(2+) influx and protein kinase C activation may be important signaling events mediating prolactin release in the goldfish.
Wong
Anderson O L
AO
Department of Zoology, University of Hong Kong, Pokfulam Road, Hong Kong, Hong Kong.
Cheung
Hilda Y S
HY
Lee
Eric K Y
EK
Chan
King Ming
KM
Cheng
Christopher H K
CH
D000818:Animals; D000001:Calcimycin / Q000494:pharmacology; D002478:Cells, Cultured; D004926:Escherichia coli / Q000235:genetics; D006054:Goldfish / Q000378:metabolism*; D007476:Ionophores / Q000494:pharmacology; D007700:Kinetics; D011388:Prolactin / Q000032:analysis / Q000096:biosynthesis* / Q000235:genetics; D011863:Radioimmunoassay / Q000379:methods / Q000662:veterinary*; D011869:Radioligand Assay / Q000379:methods / Q000662:veterinary*; D011994:Recombinant Proteins; D015203:Reproducibility of Results; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
D007476:Ionophores; D011994:Recombinant Proteins; D000001:Calcimycin; D011388:Prolactin; D013755:Tetradecanoylphorbol Acetate