Liu Guang, Yang Rui-Feng, Shi Bing-Yang, Liu De-Pei
National Laboratory of Medical Molecular Biology, Institute of Basic Medical Sciences, CAMS and PUMC, Beijing 100005, China.
Zhongguo Yi Xue Ke Xue Yuan Xue Bao. 2011 Dec;33(6):638-43.
To obtain human mitochondrial transcription factor A (TFAM), mitochondrial transcription factor B1 (TFB1M), and mitochondrial transcription factor B2 (TFB2M) that were expressed efficiently in E. coli BE21 and to purify the target proteins.
TFAM, TFB1M, and TFB2M segments were designed and synthesized. After having been sequenced, the reconstructed expression vectors were constructed by enzyme digestion and by cloning into an expression vector pET42a. Then the reconstructed vectors were transformed into E. coli BL21. Recombinant glutathione S transferase (GST) fusion proteins were expressed via the induction of IsoPropyl beta-D-ThioGalactoside (IPTG) and purified by glutathione Sepharose 4B.
The expression plasmids of pET42a-TFAM, pET42a-TFB1M, and pET42a-TFB1M were successfully constructed. The sequences of the cloned gene segments were identical with GenBank reported. The protein bands with relative molecular masses of 56 000, 67 000, and 69 000 appeared on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) after the expressed GST-TFAM, GST-TFB1M, and GST-TFB2M fusion proteins were separated by SDS-PAGE. The expressed fusion proteins were purified to high purity.
The recombinant plasmids pET42a-TFAM, pET42a-TFB1M, and pET42a-TFB2M were successfully constructed, and the GST-fused target proteins were prepared.
获得在大肠杆菌BE21中高效表达的人线粒体转录因子A（TFAM）、线粒体转录因子B1（TFB1M）和线粒体转录因子B2（TFB2M），并纯化目的蛋白。
设计并合成TFAM、TFB1M和TFB2M片段。测序后，通过酶切并克隆到表达载体pET42a中构建重组表达载体。然后将重组载体转化到大肠杆菌BL21中。通过异丙基-β-D-硫代半乳糖苷（IPTG）诱导表达重组谷胱甘肽S-转移酶（GST）融合蛋白，并通过谷胱甘肽琼脂糖4B进行纯化。
成功构建了pET42a-TFAM、pET42a-TFB1M和pET42a-TFB2M的表达质粒。克隆的基因片段序列与GenBank报道的一致。表达的GST-TFAM、GST-TFB1M和GST-TFB2M融合蛋白经十二烷基硫酸钠聚丙烯酰胺凝胶电泳（SDS-PAGE）分离后，在SDS-PAGE上出现了相对分子质量为56 000、67 000和69 000的蛋白条带。表达的融合蛋白被纯化到高纯度。
成功构建了重组质粒pET42a-TFAM、pET42a-TFB1M和pET42a-TFB2M，并制备了GST融合的目的蛋白。
