A bicistronic expression strategy for large scale expression and purification of full-length recombinant human parathyroid hormone for osteoporosis therapy

作者：Wang, Wenju; Tang, Weikun; Yan, Min; He, Kunyun; Yang, Lei; Jiang, Lin; Hua, Xiang; Yin, Li; Sun, Maosheng; Li, Hongjun^*

来源：Protein Expression and Purification, 2010, 69(2): 178-185.

DOI：10.1016/j.pep.2009.08.003

• Parathyroid hormone
• Bicistron
• Expression
• Purification
• Activity

Parathyroid hormone (PTH) contributes to the increase of trabecular connectivity and is a candidate medication for effective treating osteoporosis. PTH is a protein of 84 amino acids and some studies have suggested that the active site lies within the range from amino acid (aa) I to 34. However, a few reports have indicated a causal relationship between PTH (aa 1-34) and osteogenic sarcoma in rats, while some less obvious but important roles of the carboxyl-terminus of PTH were also found. Unfortunately, it is difficult to obtain the active integrated PTH (1-84) in vitro, due to the instability of both the protein and its mRNA. Because an alternative translation start site is located at +25 nucleotides downstream of the true start site, a truncated PTH can be translated. We constructed a rhPTH bicistronic expression plasmid (pTrepth) that could highly express non-fusion soluble rhPTH proteins in Escherichia coli. The BL-21 (DE3) containing pTrepth was cultured on a small scale until satisfactory expression and purification results were obtained. We then amplified the transformed cells in a 15-L fermentor and harvested 27 g/L cells (wet weight). Extensive rhPTH purification was achieved by a three step chromatography process. Activity tests demonstrated that our purified protein could dramatically increase cAMP in osteosarcoma cells in vitro.

• 出版日期2010-2
• 单位中国医学科学院医学生物学研究所