广东海洋大学学报Issue(1)：44-49,6.
红笛鲷tdt基因融合蛋白原核表达条件的优化及纯化
Purification and Optimization of Prokaryotic Expression of tdt Gene of Red Snapper (Lutjanus sanguineus)
摘要
Abstract
The gene sequence of coding the mature peptide Lutjanus sanguineus Terminal deoxynucleotidyl 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-β-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 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.关键词
红笛鲷/tdt基因/原核表达/优化/纯化/Western blot分析Key words
Lutjanus sanguineus/tdt gene/prokaryotic expression/optimization/purification/Western blot analysis分类
农业科技引用本文复制引用
黄瑜,张雪利,鲁义善,简纪常,吴灶和,黄浦江,周泽军..红笛鲷tdt基因融合蛋白原核表达条件的优化及纯化[J].广东海洋大学学报,2013,(1):44-49,6.基金项目
国家自然科学基金项目(41240041) （41240041）
广东省科技厅国际合作项目(2012B050600029) （2012B050600029）
