High-yield expression of fully bioactive N-terminal parathyroid hormone analog in Escherichia coli.
A fully active analog of human parathyroid hormone (hPTH) has been produced by recombinant expression in Escherichia coli. Initially, a nucleotide sequence encoding hPTH(1-34)-Asp-Pro was ligated to a proinsulin gene in the plasmid pUC8, for the eventual expression of a fusion protein of 137 amino acids. Unexpectedly, the proinsulin gene and 340 bp downstream were deleted by an unknown mechanism during transformation of the E. coli. This resulted in a new plasmid encoding a small (72-amino acid) fusion product of hPTH(1-34)-Asp35-Pro36-X, where X is a 36-residue "arbitrary" downstream sequence of pUC8. The fusion product was efficiently expressed and the hPTH analog, [Asp35]hPTH-(1-35), was readily released by acid cleavage, with a yield of 100 mg/L. This analog had an effective concentration for half-maximal adenylyl cyclase stimulation (EC50) in rat osteosarcoma cells of 14 nM, which was identical to that for hPTH-(1-34). In the ovariectomized rat model of osteoporosis, [Asp35]hPTH-(1-35) was fully active as a bone anabolic agent.
Sung
W L
WL
Institute of Biological Sciences, National Research Council, Ottawa, ON, Canada. wing.sung@nrc.ca
Chan
B S
BS
Luk
C K
CK
Zahab
D M
DM
Willick
G E
GE
Barbier
J R
JR
Isaacs
R
R
Maclean
S
S
Ross
V
V
Morley
P
P
Whitfield
J F
JF
D000262:Adenylyl Cyclases / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D004926:Escherichia coli; D005260:Female; D006801:Humans; D008969:Molecular Sequence Data; D010024:Osteoporosis / Q000517:prevention & control*; D012516:Osteosarcoma; D010052:Ovariectomy; D010957:Plasmids; D011384:Proinsulin / Q000235:genetics; D051381:Rats; D017207:Rats, Sprague-Dawley; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000494:pharmacology; D017384:Sequence Deletion; D019379:Teriparatide / Q000737:chemistry / Q000302:isolation & purification / Q000494:pharmacology*; D014407:Tumor Cells, Cultured
D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D019379:Teriparatide; D011384:Proinsulin; D000262:Adenylyl Cyclases