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Wang W.,Peking Union Medical College | Tang W.,Kunming YunJian Pharmaceutical Co. | Yan M.,Peking Union Medical College | He K.,Kunming YunJian Pharmaceutical Co. | And 6 more authors.
Protein Expression and Purification | Year: 2010

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) 1 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. © 2009 Elsevier Inc. All rights reserved.

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