Extracellular Production of Human Parathyroid Hormone as a Thioredoxin Fusion Form in Escherichia coli by Chemical Permeabilization Combined with Heat Treatment

摘要

A pET system encoding the fusion protein gene of thioredoxin (Trx) and human parathyroid hormone (hPTH) was introduced into Escherichia coli BL21 (DE3).Recombinant Trx-hPTH fusion protein was expressed in soluble form in the cytoplasm of 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 at the exponential growth phase of E.coli and by heat treatment of the culture at the end of the fermentation.A concentration of 1% (v/v) Triton X-100 was added into the culture 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 the total recombinant Trx-hPTH was released into the fermentation broth.Also,a much higher volumetric yield of recombinant Trx-hPTH could be obtained with protein release compared to yield without protein release.Simultaneously,owing to the highly thermal stability of Trx-hPTH fusion protein,heat treatment of the fermentation broth at 80 deg 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 the recombinant Trx-hPTH fusion protein into the fermentation broth but also precipitated/ denatured most of the nontarget proteins released in the broth.The tactics described herein integrated the fermentation process with subsequent recovery steps and thus provided a valuable and economical method for the production of Trx-hPTH and maybe some other Trx fusions in E.coli.