Extracellular Production of Human Parathyroid Hormone as a Thioredoxin Fusion Form in Escherichia coli by Chemical Permeabilization Combined with Heat Treatment
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- 作者：Xiang-Yang Fu ; Wang-Yu Tong ; and Dong-Zhi Wei
- 刊名：Biotechnology Progress
- 出版年：2005
- 出版时间：October 2005
- 年：2005
- 卷：21
- 期：5
- 页码：1429 - 1435
- 全文大小：245K
- 年卷期：v.21,no.5(October 2005)
- ISSN：1520-6033
文摘
A pET system encoding the fusion protein gene of thioredoxin (Trx) and humanparathyroid hormone (hPTH) was introduced into Escherichia coli BL21 (DE3).Recombinant Trx-hPTH fusion protein was expressed in soluble form in the cytoplasmof the E. coli transformant. To recover Trx-hPTH from the E. coli culture efficiently,a novel tactic was developed by adding Triton X-100 into the fermentation culture atthe exponential growth phase of E. coli and by heat treatment of the culture at theend of the fermentation. A concentration of 1% (v/v) Triton X-100 was added into theculture at the same time as IPTG addition after optimization. Under these conditions,addition of Triton X-100 had little effect on the cell growth, but more than 75% of thetotal recombinant Trx-hPTH was released into the fermentation broth. Also, a muchhigher volumetric yield of recombinant Trx-hPTH could be obtained with proteinrelease compared to yield without protein release. Simultaneously, owing to the highlythermal stability of Trx-hPTH fusion protein, heat treatment of the fermentation brothat 80 
C for 15 min at the end of fermentation was employed for primary purification.Results demonstrated that heat treatment not only boosted further release of therecombinant Trx-hPTH fusion protein into the fermentation broth but also precipitated/denatured most of the nontarget proteins released in the broth. The tactics describedherein integrated the fermentation process with subsequent recovery steps and thusprovided a valuable and economical method for the production of Trx-hPTH and maybesome other Trx fusions in E. coli.
C for 15 min at the end of fermentation was employed for primary purification.Results demonstrated that heat treatment not only boosted further release of therecombinant Trx-hPTH fusion protein into the fermentation broth but also precipitated/denatured most of the nontarget proteins released in the broth. The tactics describedherein integrated the fermentation process with subsequent recovery steps and thusprovided a valuable and economical method for the production of Trx-hPTH and maybesome other Trx fusions in E. coli.