文献类型：期刊文献

英文题名：Large scale preparation of recombinant human parathyroid hormone 1-84 from Escherichia coli

作者：Liu, Qinghai; Lin, Jinping; Liu, Meiyun; Tao, Xinyi; Wei, Dongzhi; Ma, Xingyuan; Yang, Shengli

机构：[1]E China Univ Sci & Technol, Inst Biochem, State Key Lab Bioreactor Engn, Shanghai 200237, Peoples R China;[2]Chinese Acad Sci, Shanghai Res Ctr Biotechnol, Shanghai 200233, Peoples R China

年份：2007

卷号：54

期号：2

起止页码：212

外文期刊名：PROTEIN EXPRESSION AND PURIFICATION

收录：;WOS:【SCI-EXPANDED(收录号:WOS:000247541200003)】；

语种：英文

外文关键词：rhPTH(1-84); fusion protein; chromatography; enterokinase; adenylate cyclase; large scale preparation; Escherichia coli

摘要：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 300 mg/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. (C) 2007 Elsevier Inc. All rights reserved.