Abstract: To efficiently prepare soluble human-derived Nanog protein and reduce the cost, recombinant expression Escherichia coli(pET32a-Nanog) was constructed by molecular cloning technology and soluble fusion expression was induced by isopropyl-β-D-thiogalactopyranoside(IPTG). The product was purified by Ni-NTA affinity chromatography and was digested by enterokinase to obtain the target protein. Western blot was used to identify the specificity of antibody binding to the protein and the fermentation conditions were further optimized. The results showed that the prokaryotic expression vector pET32a-Nanog was successfully constructed and could be fused expression with the thioredoxin in E.coli. The fusion protein was digested by enterokinase to obtain a protein with a relative molecular mass of about 38 kDa. The protein could bind to Nanog antibodies specifically by Western blot. The optimal fermentation results under shake culture conditions contained induction temperature of 37 ℃, IPTG concentration 0.2 mmol/L, glycerin as the feed carbon source. The dissolved oxygen and temperature control methods were further optimized in a 15 L fermenter. The highest Nanog yield was up to 1 386.4 mg/L after 12 hours with the dissolved oxygen set at 30%, 37 ℃(30 ℃ for 30 min during the induction period), and reached 90% purity after protein purification. This study provided the base for the subsequent preparation of Nanog polyclonal antibodies and Nanog basic medical experimental research.
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