Full-length chicken parathyroid hormone. Biosynthesis in Escherichia coli and analysis of biologic activity.
Chicken parathyroid hormone (cPTH) has been reported to stimulate adrenal steroidogenesis and to have unusual potency on traditional PTH target tissues. To evaluate these properties, chicken PTH-(1-88) has been expressed in Escherichia coli using a plasmid encoding a fusion protein which links together growth hormone, a factor Xa recognition site, and chicken PTH-(1-88). The growth hormone-cPTH fusion protein required the presence of 0.02% sodium dodecyl sulfate to remain in solution and be cleaved by factor Xa. The high performance liquid chromatography-purified recombinant cPTH-(1-88) and chemically synthesized cPTH-(1-34) had similar potency in rat osteosarcoma (ROS 17/2.8) cells, opossum kidney (OK) cells, and dispersed primary chicken kidney cells. The biologic potencies of cPTH-(1-34) and cPTH-(1-88) in radioreceptor binding and cAMP generation in both bone- and kidney-derived cell lines were less than those of human (h)PTH-(1-34). In dispersed chicken kidney cells, cAMP production by cPTH-(1-34) and cPTH-(1-88) was similar to that stimulated by human PTH-(1-34). No stimulation of steroidogenesis could be detected when recombinant chicken PTH-(1-88) was added to dispersed chicken adrenal cells. The biologic activity of recombinant chicken PTH-(1-88) purified from E. coli was comparable with that of chicken PTH-(1-88) expressed by mammalian COS cells. Thus, the full-length chicken PTH did not exhibit enhanced potency, when compared with human PTH in ROS 17/2.8, OK cell lines, and dispersed chicken kidney cells and did not demonstrate the novel steroidogenic action previously reported in adrenal cells. The successful production of chicken PTH-(1-88) will enhance our understanding of the structure-activity relationships for PTH, particularly the sequence-dependent metabolism of the hormone.
Lim
S K
SK
Endocrine Unit, Massachusetts General Hospital, Boston.
Gardella
T
T
Thompson
A
A
Rosenberg
J
J
Keutmann
H
H
Potts
J
J
Kronenberg
H
H
Nussbaum
S
S
D000818:Animals; D002645:Chickens; D002851:Chromatography, High Pressure Liquid; D000242:Cyclic AMP / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics*; D015951:Factor Xa / Q000494:pharmacology; D015964:Gene Expression Regulation, Bacterial; D005798:Genes, Bacterial; D007150:Immunohistochemistry; D010281:Parathyroid Hormone / Q000096:biosynthesis* / Q000302:isolation & purification / Q000378:metabolism; D010957:Plasmids; D011869:Radioligand Assay; D051381:Rats; D014407:Tumor Cells, Cultured
D010281:Parathyroid Hormone; D000242:Cyclic AMP; D015951:Factor Xa