Partial purification of human parathyroid hormone 1-84 as a thioredoxin fusion form in recombinant Escherichia coli by thermoosmotic shock.
A modified purification method, thermoosmotic shock (osmotic shock coupled with heat-treatment) for heat-stable proteins, was devised in the purification of Trx-hPTH (1-84) (human parathyroid hormone coupled with thioredoxin as a fusion partner) from E. coli. Thermoosmotic shock can integrate the functions of extraction and crude separation of fusion protein Trx-hPTH (1-84). To improve the purification efficiency, thermoosmotic shock conditions were optimized and achieved as follows: the optimized high osmotic solution containing 20mM Tris-HCl buffer (pH 8.0), 1mM EDTA, and 25% sucrose; the low osmotic solution containing 20mM Tris-HCl buffer (pH 8.0), 1mM EDTA, and the heat-treatment temperature of 100 degrees C for 10 min. Using this method, the purity of Trx-hPTH (1-84) was up to 73% and the yield was up to 72%, respectively. In addition, the two separation methods of both thermoosmotic shock and affinity chromatography have been compared, indicating that thermoosmotic shock is an economical and feasible way for the fusion protein separation. Besides, the thermoosmotic shock method may be used for the purification of some proteins of thermal stability without N-terminal His-tag.
Guo
Qi-Ran
QR
State Key Laboratory of Bioreactor Engineering, Institute of New World Biotechnology, East China University of Science and Technology, Shanghai 200237, China.
Wei
Dong-Zhi
DZ
Tong
Wang-Yu
WY
D002846:Chromatography, Affinity; D000242:Cyclic AMP / Q000378:metabolism; D004492:Edetic Acid; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D009997:Osmotic Pressure; D010281:Parathyroid Hormone / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism* / Q000494:pharmacology; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism* / Q000494:pharmacology; D013696:Temperature; D013879:Thioredoxins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*
C470125:PTH protein, human; D010281:Parathyroid Hormone; D011993:Recombinant Fusion Proteins; D013879:Thioredoxins; D004492:Edetic Acid; D000242:Cyclic AMP