Extracellular production of human parathyroid hormone as a thioredoxin fusion form in Escherichia coli by chemical permeabilization combined with heat treatment.
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 degrees 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.
Fu
Xiang-Yang
XY
State Key Laboratory of Bioreactor Engineering, Institute of New World Biotechnology, East China University of Science and Technology, Shanghai 200237, China.
Tong
Wang-Yu
WY
Wei
Dong-Zhi
DZ
D019149:Bioreactors / Q000382:microbiology*; D018929:Cell Culture Techniques / Q000379:methods*; D005591:Chemical Fractionation / Q000379:methods*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D045604:Extracellular Fluid / Q000378:metabolism; D006358:Hot Temperature; D006801:Humans; D010281:Parathyroid Hormone / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D010539:Permeability; D015202:Protein Engineering / Q000379:methods*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000302:isolation & purification; D013879:Thioredoxins / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification
D010281:Parathyroid Hormone; D011993:Recombinant Fusion Proteins; D013879:Thioredoxins