CAJ | 학술논문

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.
극륭편마홍적조(Lutjanus sanguineus)말단탈양핵당핵산전이매 TdT 단백(Terminal deoxynucleotidyl transferases)성숙태기인서렬,병여 pET-32a(+)재체련접,구건원핵표체재체 pET-32a-TdT,재장기전입대장간균BL21(DE3)균주,이용이병기-β-D-류대반유당감(IPTG)진행유도표체.운용전통방법우화유도조건,이제고중조융합단백적표체효솔.SDS-PAGE분석표명,37℃、0.7 mmol/L IPTG조건하유도4 h 후,TdT융합단백적표체량최대,분자질량대소여예측치상부,해단백주요이포함체형식존재.이용 HisTrap HP친화층석주사TdT단백득도진일보순화,최가미서세탈농도위300 mmol/L,Western blot분석현시,해융합단백가여서항His-tag단극륭항체발생특이성적결합,표명표체단백위목적단백.
@@@@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.