Sung W L, Chan B S, Luk C K, Zahab D M, Willick G E, Barbier J R, Isaacs R, Maclean S, Ross V, Morley P, Whitfield J F
Institute of Biological Sciences, National Research Council, Ottawa, ON, Canada.
IUBMB Life. 2000 Feb;49(2):131-5. doi: 10.1080/15216540050022458.
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.
通过在大肠杆菌中进行重组表达，已产生了一种完全活性的人甲状旁腺激素（hPTH）类似物。最初，将编码hPTH(1 - 34)-Asp-Pro的核苷酸序列连接到质粒pUC8中的胰岛素原基因上，以最终表达一种137个氨基酸的融合蛋白。出乎意料的是，在大肠杆菌转化过程中，胰岛素原基因及其下游340 bp被一种未知机制删除。这产生了一种新的质粒，编码hPTH(1 - 34)-Asp35-Pro36-X的小（72个氨基酸）融合产物，其中X是pUC8的36个残基的“任意”下游序列。融合产物高效表达，并且hPTH类似物[Asp35]hPTH-(1 - 35)通过酸裂解很容易释放出来，产量为100 mg/L。该类似物在大鼠骨肉瘤细胞中对腺苷酸环化酶刺激的半最大有效浓度（EC50）为14 nM，与hPTH-(1 - 34)相同。在去卵巢大鼠骨质疏松模型中，[Asp35]hPTH-(1 - 35)作为一种骨合成代谢剂具有完全活性。
