Large scale preparation of recombinant human parathyroid hormone 1-84 from Escherichia coli.
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.
Liu
Qinghai
Q
State Key Laboratory of Bioreactor Engineering, Institute of Biochemistry, East China University of Science and Technology, Shanghai 200237, PR China.
Lin
Jinping
J
Liu
Meiyun
M
Tao
Xinyi
X
Wei
Dongzhi
D
Ma
Xingyuan
X
Yang
Shengli
S
D000262:Adenylyl Cyclases / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001681:Biological Assay; D002850:Chromatography, Gel; D004765:Enteropeptidase / Q000378:metabolism; D004926:Escherichia coli / Q000378:metabolism; D006801:Humans; D012516:Osteosarcoma / Q000201:enzymology; D010281:Parathyroid Hormone / Q000302:isolation & purification* / Q000378:metabolism / Q000494:pharmacology; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification / Q000378:metabolism / Q000494:pharmacology
C470125:PTH protein, human; D010281:Parathyroid Hormone; D011993:Recombinant Fusion Proteins; D004765:Enteropeptidase; D000262:Adenylyl Cyclases