重组人甲状旁腺激素(1-34)在大肠杆菌中的表达与纯化

将化学合成的rhPTH(1-34)基因用PCR扩增后,克隆至表达载体pET-35b( ),使rhPTH(1—34)融合于纤维素结合结构域(CBDdos)的羧基端,并得到高效表达．融合蛋白经纤维素树脂亲和层析纯化后,经Factor Xa裂解释放出rhPTH(1-34),再通过纤维素树脂亲和层析、C4反向高效液相色谱纯化得到rhPTH(1-34)纯品．每升培养液可获取3mg高纯度的rhPTH(1-34)．经质谱测定,所得样品的分子量为4117．0Da,与rhPTH(1—34)理论分子量一致．

Expression and Purification of Recombinant Human Parathyroid Hormone (1-34) in E.coli

Parathyroid hormone is synthesized and secreted as a linear polypeptide by chief cells of parathyroid glands. The 25 N terminal amino acids of the 115 amino acid precursor, prepro parathyroid hormone, is cleaved in rough endoplasmic reticulum to form pro parathyroid hormone. Then the N terminal 6 amino acids of prepro parathyroid hormone is removed in Golgi apparatus to release parathyroid hormone of 84 amino acids. Parathyroid hormone is the most important endocrine regulator of serum calcium concentration. The decrease in serum calcium concentration directly stimulates secretion of parathyroid hormone. The action of parathyroid hormone involves the following three processes: (1) Mobilization of calcium from bones; (2) Enhanced absorption of calcium from the small intestine; (3) Suppression of calcium loss in urine. The N terminal fragment(1?34 amino acids) of parathyroid hormone is fully active for its in vivo function. In this report, we expressed recombinant human parathyroid hormone (1?34) in E.coli. The recombinant human parathyroid hormone (1?34) gene was PCR amplified by using synthetic oligonucleotides encoding recombinant human parathyroid hormone (1?34) as the template, and then cloned into pET?35b(+) vector which allowed the overexpression of recombinant human parathyroid hormone (1?34) as a C?terminal fusion of cellulose binding domain (CBDclos). The fusion protein was purified by an affinity chromatography cellulose resin and cleaved by Factor Xa to release recombinant human parathyroid hormone (1?34). After Factor Xa cleavage and purification by another affinity chromatography cellulose resin and a C4 RP?HPLC, highly purified recombinant human parathyroid hormone (1?34) was obtained at a yield of 3 mg/L. By using the mass spectrometry, the molecule weight of the product was determined to be (4 117.0) dalton, which corresponded to recombinant human parathyroid hormone (1?34).