Liu Qinghai, Lin Jinping, Liu Meiyun, Tao Xinyi, Wei Dongzhi, Ma Xingyuan, Yang Shengli
State Key Laboratory of Bioreactor Engineering, Institute of Biochemistry, East China University of Science and Technology, Shanghai 200237, PR China.
Protein Expr Purif. 2007 Aug;54(2):212-9. doi: 10.1016/j.pep.2007.03.009. Epub 2007 Mar 21.
Human parathyroid hormone (hPTH) is a promising agent in the treatment of osteoporosis. The intact recombinant human parathyroid hormone [rhPTH(1-84)] was prepared in a large scale from Escherichia coli using a soluble fusion protein strategy. With degenerate codons, gene of hPTH(1-84) was synthesized, ligated with pET32a(+) vector, and then expressed in E. coli BL21(DE3) cells. The soluble fusion protein His(6)-thioredoxin-hPTH(1-84) was harvested after purification by immobilized metal affinity chromatography (IMAC). Following enterokinase cleavage, ion-exchange-chromatography (IEC) and size-exclusive-chromatography (SEC) were used, and finally, over 300mg/l intact hPTH(1-84) with high purity up to 99% was obtained. The purified rhPTH(1-84) was confirmed by mass spectrometry and N-terminal/C-terminal amino-acid sequence analysis. Additionally, this product stimulated adenylate cyclase in Rat Osteosarcoma Cell UMR-106 at the same extent as hPTH standards, indicating that the purified rhPTH(1-84) has full biological activity. The efficient procedure for expression and purification of rhPTH(1-84) may be useful for the mass production of this important protein.
人甲状旁腺激素（hPTH）是治疗骨质疏松症的一种很有前景的药物。完整的重组人甲状旁腺激素[rhPTH(1-84)]采用可溶性融合蛋白策略在大肠杆菌中大规模制备。使用简并密码子合成hPTH(1-84)基因，将其与pET32a(+)载体连接，然后在大肠杆菌BL21(DE3)细胞中表达。通过固定化金属亲和色谱（IMAC）纯化后收获可溶性融合蛋白His(6)-硫氧还蛋白-hPTH(1-84)。经肠激酶切割后，使用离子交换色谱（IEC）和尺寸排阻色谱（SEC），最终获得了超过300mg/l、纯度高达99%的完整hPTH(1-84)。通过质谱和N端/C端氨基酸序列分析对纯化的rhPTH(1-84)进行了确认。此外，该产品在大鼠骨肉瘤细胞UMR-106中刺激腺苷酸环化酶的程度与hPTH标准品相同，表明纯化的rhPTH(1-84)具有完全的生物活性。rhPTH(1-84)高效的表达和纯化方法可能有助于这种重要蛋白质的大规模生产。
