Liu Guo-Dan, Zhou Shi-Feng, Ding Xu-Chen, Fang Chun-Lai, Mi Shu-Yong, Gao Xiang-Chun, Han Qing
Department of Ophthalmology, the Fourth Affiliated Hospital of Harbin Medical University, Harbin 150001, Heilongjiang Province, China.
Department of Emergency, the First Affiliated Hospital of Harbin Medical University, Harbin 150001, Heilongjiang Province, China.
Int J Ophthalmol. 2017 Apr 18;10(4):560-566. doi: 10.18240/ijo.2017.04.10. eCollection 2017.
To develop a new method to produce recombinant reprogramming proteins, cMyc, Klf4, Oct4, and Sox2, in soluble format with low cost for the generation of induced pluripotent stem cells (iPSCs).
A short polypeptide sequence derived from the HIV trans-activator of transcription protein (TAT) and the nucleus localization signal (NLS) polypeptide were fused to the N terminus of the reprogramming proteins and they were constructed into pCold-SUMO vector which can extremely improve the solubility of recombinant proteins. Then these vector plasmids were transformed into BL21 (DE3) Chaperone competent cells for amplification. The solubility of these recombinant proteins was determined by SDS-PAGE and Coomassie brilliant blue staining. The recombinant proteins were purified by Ni-NTA resin and identified by Western blot. The transduction of these proteins into HEK 293T cells were evaluated by immunofluorescence staining.
These four reprogramming proteins could be produced in soluble format in pCold-SUMO expression vector system with the assistance of chaperone proteins in bacteria. The proteins were purified successfully with a purity of over 70% with a relative high transduction rate into 293 cells.
The results in the present study indicate the four important reprogramming proteins, cMyc, Klf4, Oct4, and Sox2, can be produced in soluble format in bacteria with low cost. Our new method thus might be expected to greatly contribute to the future study of iPSCs.
开发一种新方法，以低成本生产可溶性的重组重编程蛋白cMyc、Klf4、Oct4和Sox2，用于诱导多能干细胞（iPSC）的生成。
将源自HIV转录反式激活蛋白（TAT）的短多肽序列和核定位信号（NLS）多肽与重编程蛋白的N端融合，并构建到能极大提高重组蛋白溶解度的pCold-SUMO载体中。然后将这些载体质粒转化到BL21（DE3）伴侣感受态细胞中进行扩增。通过SDS-PAGE和考马斯亮蓝染色测定这些重组蛋白的溶解度。用Ni-NTA树脂纯化重组蛋白，并通过蛋白质免疫印迹法进行鉴定。通过免疫荧光染色评估这些蛋白导入HEK 293T细胞的情况。
在细菌中伴侣蛋白的协助下，这四种重编程蛋白可以在pCold-SUMO表达载体系统中以可溶性形式产生。这些蛋白成功纯化，纯度超过70%，导入293细胞的转导率相对较高。
本研究结果表明，四种重要的重编程蛋白cMyc、Klf4、Oct4和Sox2可以在细菌中以低成本可溶性形式产生。因此，我们的新方法有望对iPSC的未来研究做出巨大贡献。
