Expression of human parathyroid hormone-(1-84) in Escherichia coli as a factor X-cleavable fusion protein.
Recombinant human parathyroid hormone (hPTH)-(1-84) was obtained from Escherichia coli using a cleavable fusion protein strategy. The fusion protein contains residues 1-138 of human growth hormone as the amino-terminal region and residues 1-84 of hPTH as the carboxyl-terminal region. A 7-residue linker containing the recognition/cleavage sequence of the site-specific blood coagulation protease activated factor X (factor Xa) joins the two regions. Intact hPTH-(1-84) is released from this fusion protein by cleavage in vitro with factor Xa. The fusion protein was produced at a high level and formed inclusion bodies which allowed it to be easily purified by low speed centrifugation, with a yield of approximately 50 mg/liter of culture. After factor Xa cleavage and high performance liquid chromatography purification, highly purified hPTH was obtained, with a final yield of 1.5-3 mg/liter. Physical and biological characterization of the purified hormone demonstrated that it was intact and active hPTH-(1-84).
Gardella
T J
TJ
Endocrine Unit, Massachusetts General Hospital, Boston.
Rubin
D
D
Abou-Samra
A B
AB
Keutmann
H T
HT
Potts
J T
JT
Kronenberg
H M
HM
Nussbaum
S R
SR
D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D002851:Chromatography, High Pressure Liquid; D000242:Cyclic AMP / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics*; D005170:Factor X / Q000378:metabolism*; D015870:Gene Expression; D006801:Humans; D008969:Molecular Sequence Data; D008970:Molecular Weight; D012516:Osteosarcoma; D010281:Parathyroid Hormone / Q000235:genetics* / Q000302:isolation & purification / Q000494:pharmacology; D010957:Plasmids; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification / Q000494:pharmacology; D015183:Restriction Mapping
D010281:Parathyroid Hormone; D011993:Recombinant Fusion Proteins; D005170:Factor X; D000242:Cyclic AMP