九香虫CcPT1蛋白的原核表达及纯化

Prokaryotic expression and purification of CcPT1 protein from Aspongopus chinensis

目的 原核表达九香虫Cc PT1蛋白,并进行纯化。方法 将合成的Cc PT1基因克隆至载体p GEX-4T-1,构建重组表达质粒p GEX-4T1-Cc PT1,转化感受态E.coli Rosetta,经IPTG诱导表达重组蛋白,并优化诱导表达的温度(20及37℃)、IPTG终浓度(0.25、0.5、0.75、1 mmol/L)及时间(6、8、10、12 h)。采用GST蛋白纯化系统纯化重组蛋白,纯化产物经10%SDS-PAGE分析及Western blot鉴定,纯化过程中采用Pre Scission Protease去除GST标签。结果 重组蛋白GST-Cc PT1相对分子质量约为29 800,大小与预期相符,主要以包涵体形式表达,蛋白浓度为0.026 9 mg/m L。最适诱导条件为:20℃下,采用终浓度0.75 mol/L的IPTG诱导12 h。纯化蛋白纯度> 90%,且可与小鼠抗GST单克隆抗体发生特异性结合。去除GST标签后的Cc PT1蛋白相对分子质量约为2 830,蛋白得率达11.15%。结论 经原核表达获得了纯度较高的九香虫Cc PT1蛋白,为九香虫抗癌肽的深入研究奠定了基础。

Objective To express and purify Cc PT1 protein from Aspongopus chinensis in prokaryotic cell.Methods Thesynthesized Cc PT1 gene was cloned to vector p GEX-4T-1 to construct recombinant expression plasmid p GEX-4T1-Cc PT1,which was then transformed to competent E.coli Rosetta strain and induced by IPTG.The induction temperature(20 ℃ and37 ℃),final concentration of IPTG(0.25,0.5,0.75 and 1 mmol/L)and induction time(6,8,10,12 h)were opti-mized.The obtained protein was purified by GST protein purification system,which was then analyzed by 10% SDS-PAGEand identified by Western blot.GST tags were removed by Pre Scission Protease during purification.Results The recombi-nant protein GST-Cc PT1 was expressed in the form of inclusion body with a concentration of 0.026 9 mg/ml,of which therelative molecular mass was 29 800,consistent with the expectation.The optimum induction condition was induction withIPTG of final concentration of 0.75 mol/L for 12 h at 20 ℃.The purified protein was more than 90% in purity and boundspecifically to mouse monoclonal antibody against GST.After remove of GST tags,Cc PT1 protein showed a relative molecu-lar mass of about 2 830 and the yield was 11.15%.Conclusion A.chinensis Cc PT1 protein was expressed by prokaryoticexpression system,and the purity of Cc PT1 protein was high after purification,which laid a foundation of the in-depth studyof anticancer peptides of A.chinensis.