Shi Hai-Yan, Li Yong-Jun, Gao Ji-Min
Zhonghua Shi Yan He Lin Chuang Bing Du Xue Za Zhi. 2013 Oct;27(5):354-6.
Express and purify four single-stranded DNA-binding (SSB) proteins, and evaluate the binding of SSB proteins on HCV RNA.
The expression plasmids of four SSB proteins were conducted, termed TTH, SSOB, KOD and BL21, respectively. The BL21 (DE3) was transformed by the expression plasmid of TTH, Transetta (DE3) were transformed by the expression plasmid of SSOB, KOD and BL21, then protein expression was induced with IPTG, the expression products were analysised by SDS-PAGE. To evaluate the binding of SSB on HCV RNA, RNA-SSB protein complexes were applied to a 1.2% TAE agarose gel.
Suitable competent cells were transformed with the expression plasmids, induced by IPTG. SSB proteins were purified by affinity chromatography, to visualize their purity all SSB proteins were applied to SDS-PAGE analysis. All four proteins showed single clear bands. We have successfully obtained the SSB protein expression plasmid, expressed and purified SSB protein. TAE agarose gel electrophoresis was used to confirm SSB protein-RNA binding activity. The each of SSB-RNA complex migrated more slowly than the sole RNA, which suggested SSB protein could specifically bind to RNA.
We have expressed and purified four SSB proteins, and for the first time found that SSB protein can bind HCV RNA. Our results may provide a basis for future studies of the novel functions of SSB proteins on RNA.
表达并纯化四种单链DNA结合（SSB）蛋白，并评估SSB蛋白与丙型肝炎病毒（HCV）RNA的结合情况。
构建四种SSB蛋白的表达质粒，分别命名为TTH、SSOB、KOD和BL21。用TTH表达质粒转化BL21（DE3），用SSOB、KOD和BL21表达质粒转化Transetta（DE3），然后用异丙基-β-D-硫代半乳糖苷（IPTG）诱导蛋白表达，通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）分析表达产物。为评估SSB与HCV RNA的结合情况，将RNA-SSB蛋白复合物应用于1.2%的TAE琼脂糖凝胶。
用表达质粒转化合适的感受态细胞，并用IPTG诱导。通过亲和层析纯化SSB蛋白，将所有SSB蛋白进行SDS-PAGE分析以观察其纯度。所有四种蛋白均显示出单一清晰条带。我们成功获得了SSB蛋白表达质粒，表达并纯化了SSB蛋白。用TAE琼脂糖凝胶电泳确认SSB蛋白与RNA的结合活性。每个SSB-RNA复合物的迁移速度均比单独的RNA慢，这表明SSB蛋白可特异性结合RNA。
我们表达并纯化了四种SSB蛋白，首次发现SSB蛋白可结合HCV RNA。我们的结果可能为未来研究SSB蛋白在RNA上的新功能提供基础。
