Recombinant human parathyroid hormone synthesized in Escherichia coli. Purification and characterization.
Recombinant human parathyroid hormone (hPTH) was expressed in Escherichia coli harboring a plasmid containing a synthetic human parathyroid hormone gene under the control of the E. coli lac promoter. Three major forms of the hormone were isolated by acid extraction and purified to homogeneity by high performance liquid chromatography. By amino acid analysis and NH2-terminal sequencing, these were identified as hPTH-(1-84), formyl-methionyl-hPTH-(1-84), and hPTH-(8-84). The recombinant hPTH-(1-84) was immunologically indistinguishable from a World Health Organization standard of extracted native hPTH-(1-84). Recombinant hPTH-(1-84) was also bioactive in renal and skeletal adenylate cyclase assays. In the skeletal bioassay performed in UMR 108 osteosarcoma cells its activity was identical to that of an hPTH-(1-84) standard. In this bioassay, formyl-methionyl-hPTH-(1-84) had 10% of the activity of hPTH-(1-84) and hPTH-(8-84) was inactive. The results demonstrate the importance of isolating hPTH-(1-84) from other recombinant forms and metabolites to achieve full hormonal bioactivity and indicate that purified recombinant hPTH-(1-84) can thereby be obtained which should be a useful source of hormone for both basic and clinical studies.
Rabbani
S A
SA
Department of Medicine, McGill University, Montreal, Quebec, Canada.
Yasuda
T
T
Bennett
H P
HP
Sung
W L
WL
Zahab
D M
DM
Tam
C S
CS
Goltzman
D
D
Hendy
G N
GN
D000262:Adenylyl Cyclases / Q000378:metabolism; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D001483:Base Sequence; D001681:Biological Assay; D001842:Bone and Bones / Q000201:enzymology; D002851:Chromatography, High Pressure Liquid; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D007668:Kidney / Q000201:enzymology; D008722:Methods; D010281:Parathyroid Hormone / Q000096:biosynthesis* / Q000302:isolation & purification; D010446:Peptide Fragments / Q000302:isolation & purification; D010957:Plasmids; D051381:Rats; D011994:Recombinant Proteins / Q000096:biosynthesis*; D014407:Tumor Cells, Cultured / Q000187:drug effects
D000596:Amino Acids; D010281:Parathyroid Hormone; D010446:Peptide Fragments; D011994:Recombinant Proteins; C054602:parathyroid hormone (8-84); C054601:formyl-methionyl-hPTH (1-84); D000262:Adenylyl Cyclases