A bicistronic expression strategy for large scale expression and purification of full-length recombinant human parathyroid hormone for osteoporosis therapy.
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 27g/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.
Wang
Wenju
W
Institute of Medical Biology, Chinese Academy of Medical Sciences, Peking Union Medical College, Kunming, People's Republic of China.
Tang
Weikun
W
Yan
Min
M
He
Kunyun
K
Yang
Lei
L
Jiang
Lin
L
Hua
Xiang
X
Yin
Li
L
Sun
Maosheng
M
Li
Hongjun
H
D000818:Animals; D045744:Cell Line, Tumor; D000242:Cyclic AMP / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005260:Female; D006801:Humans; D007118:Immunoassay / Q000379:methods; D008969:Molecular Sequence Data; D010024:Osteoporosis / Q000188:drug therapy*; D012516:Osteosarcoma / Q000378:metabolism; D010281:Parathyroid Hormone / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism / Q000627:therapeutic use*; D051381:Rats; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism / Q000627:therapeutic use*
D010281:Parathyroid Hormone; D011994:Recombinant Proteins; D000242:Cyclic AMP