摘要
克隆编码红笛鲷(Lutjanus sanguineus)末端脱氧核糖核酸转移酶TdT蛋白(Terminal deoxynucleotidyl transferases)成熟肽基因序列,并与pET-32a(+)载体连接,构建原核表达载体pET-32a-TdT,再将其转入大肠杆菌BL21(DE3)菌株,利用异丙基-β-D-硫代半乳糖苷(IPTG)进行诱导表达。运用传统方法优化诱导条件,以提高重组融合蛋白的表达效率。SDS-PAGE分析表明,37℃、0.7 mmol/L IPTG条件下诱导4 h后,TdT融合蛋白的表达量最大,分子质量大小与预测值相符,该蛋白主要以包涵体形式存在。利用HisTrap HP亲和层析柱使TdT蛋白得到进一步纯化,最佳咪唑洗脱浓度为300 mmol/L,Western blot分析显示,该融合蛋白可与鼠抗His-tag单克隆抗体发生特异性的结合,表明表达蛋白为目的蛋白。
The gene sequence of coding the mature peptide Lutjanus sanguineus Terminal deoxynueleotidyl transferases （TdT） protein was cloned and then inserted into the pET-32a（＋） vector to construct prokaryotic expression plasmid pET-32a-TdT. And then it was transferred into E. coli BL21 （DE3） strain. The recombinant TdT fusion protein was over expressed in Escherichia coli BL21（DE3） cells in the presence of isopropyl-^-thiogalactopyranoside （IPTG）. To achieve a high level expression, the optimized induction conditions were identified by using classical experimental method. SDS-PAGE analysis revealed that inducing the cells, the optimal conditions were at 37 ~C in 0.7 mmol/L IPTG for 4 hours for expression of the recombinant TdT fusion protein. The molecular mass of the expressed product was identical to the predicted protein which was mainly detected in the insoluble fraction of E. coli cell lysates. The recombinant TdT fusion protein was purified by using His Trap HP affinity column and the best elution concentration of imidazole was 300 mmol/L. Western blot analysis showed that the recombinant TdT fusion protein could be combined with mouse anti-His-Tag Mab, So the expressed protein was definitely confirmed to the aim protein.
出处
《广东海洋大学学报》
CAS
2013年第1期44-49,共6页
Journal of Guangdong Ocean University
基金
国家自然科学基金项目(41240041)
广东省科技厅国际合作项目(2012B050600029)
