细胞穿透性重组酶Tat-FLPo的表达、纯化及活性检测

中国生物工程杂志

2013, Vol. 33

Issue (8): 84-90

技术与方法

细胞穿透性重组酶Tat-FLPo的表达、纯化及活性检测

王琪^1,2, 俞慧清¹, 陈建泉¹, 曾宪垠², 成国祥¹

1. 上海转基因研究中心上海 201210;
2. 四川农业大学雅安 625014

The Optimization,Purification and Activity Detection of the Cell Penetrating Recombinase Tat-FLPo

WANG Qi^1,2, YU Hui-qing¹, CHEN Jian-quan¹, ZENG Xian-yin², CHENG Guo-xiang¹

1. Shanghai Transgenic Research Center, Shanghai 201210, China;
2. Atomic Energy Application Laboratory, Sichuan Agriculture University, Ya’an 625014, China

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摘要： 为了获得细胞穿透性的重组酶Tat-FLPe应用于基因重组研究,建立了高效稳定的Tat-FLPe原核表达与纯化体系。首先,以质粒pCAGGS-FLPe为模板,PCR扩增 FLPe序列,将其克隆到pET28a-Tat中并与穿膜蛋白Tat相连,获得表达载体pET28a-Tat-FLPe,然后将Tat-FLPe分别插入到另外3个原核表达载体pET22b、pET30a、pET32a中,转入原核表达菌Rosetta诱导表达,结果发现只有载体pET32a-Tat-FLPe可在大肠杆菌中表达出稳定的、有活性的Tat-FLPe;在此基础上,为进一步提高Tat-FLPe的表达量,利用在线软件对Tat-FLPe的密码子进行了优化,发现优化后 Tat-FLPe的表达量显著提高;另一方面还探索了诱导表达条件对Tat-FLPe表达的影响,发现Tat-FLPe转化菌的最佳诱导表达条件为0.05mol/L IPTG,30℃诱导表达4h。最后,采用阳离子交换柱纯化表达上清,获得细胞穿透性的Tat-FLPe,并通过质粒酶切实验及细胞实验验证了Tat-FLPe的体内体外活性。成功地在原核表达系统中获得了有活性的Tat-FLPe,并优化了其表达体系,为下一步应用于细胞及活体动物的基因操作奠定了基础。

关键词： Tat-FLPe; 原核表达; 优化; 纯化

Abstract: In order to obtain the cell permeable recombinase Tat-FLPe for recombinant DNA research, a highly efficient and stable Tat-FLPe prokaryotic expression and purification system was established. First, the FLPe sequence was amplified by using the plasmid pCAGGS-FLPe as template, and then inserted FLPe into prokaryotic expression vector pET28a-Tat to get vector pET28a-Tat-FLPe for expression. Tat-FLPe were cloned into another three prokaryotic expression vector pET22b, pET30a, pET32a for expression respectively. As a result, only vector pET32a-Tat-FLPe induced stable Tat-FLPe expression in transformed Rosetta cells, while other three vectors failed to express or express at trace level. Further, in order to improve the expression level of Tat-FLPe, the composed codon of FLPe was optimized using online software. The expression level of optimized Tat-FLPe was increased significantly compared to the original one. On the other hand, the inducible conditions which could affect Tat-FLPe expression were explored, and found that the optimal induction condition of transformed cells was 0.05mol/L IPTG, 30 ℃, incubated for 4 hours. Finally, the expressed Tat-FLPe in Rosetta cells was purified by cation exchange column, the activity of cell permeable TAT-FLPe was verified by plasmid digestion experiments in vitro and cell experiments in vivo. In summary, the biological active Tat-FLPe recombinase in prokaryotic expression system were expressed successfully, thus laid a sound foundation for its application in genetic manipulation of cells and living animals.

Key words: Tat-FLPe Prokaryotic expression Optimization Purification

收稿日期: 2013-04-15 出版日期: 2013-08-25

ZTFLH:

Q786

基金资助: 国家转基因生物新品种培育重大专项资助项目(2011ZX08008-004,2009ZX08008-009B)

通讯作者: 俞慧清,E-mail:yhq202@hotmail.com E-mail: yhq202@hotmail.com

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引用本文:

王琪, 俞慧清, 陈建泉, 曾宪垠, 成国祥. 细胞穿透性重组酶Tat-FLPo的表达、纯化及活性检测[J]. 中国生物工程杂志, 2013, 33(8): 84-90.

WANG Qi, YU Hui-qing, CHEN Jian-quan, ZENG Xian-yin, CHENG Guo-xiang. The Optimization,Purification and Activity Detection of the Cell Penetrating Recombinase Tat-FLPo. China Biotechnology, 2013, 33(8): 84-90.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/ 或 https://manu60.magtech.com.cn/biotech/CN/Y2013/V33/I8/84

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