牛mettl3基因的原核表达与蛋白纯化

Prokaryotic expression and protein purification of dairy cow mettl3 gene

m^6A甲基转移酶(methyltransferase-like protein 3,METTL3)可调控m RNA的m6A甲基化水平,调节细胞蛋白质合成,实验室前期发现METTL3为乳腺上皮细胞乳合成重要调节分子,发挥泌乳调节作用。目前尚不清楚METTL3分子结构基础及其参与的调节机制。为揭示METTL3结构与功能关系,对METTL3蛋白作原核表达及纯化。研究采用PCR方法扩增牛mettl3基因完整CDS区,将CDS区插入原核表达载体p GEX-4T-1,异丙基硫代-β-D-半乳糖苷(IPTG)诱导METTL3蛋白大量表达,用8 mol·L-1尿素裂解包涵体,利用GST蛋白纯化试剂盒纯化诱导获得METTL3蛋白。SDS-PAGE证实GST-METTL3融合蛋白存在于包涵体内,试验获得高纯度GST-METTL3融合蛋白,为进一步研究METTL3蛋白在奶牛泌乳中作用提供重要依据。

m^6A methyltransferase（methyltransferase-like protein 3, METTL3） can regulate m6 A methylation levels in m RNA, thus plays an important role in regulating protein synthesis in cells. The previous laboratory study found that METTL3 was one of the important regulatory molecules for milk synthesis in bovine mammary epithelial cells, regulating the lactation. It was still unknown the molecular basis and regulation mechanism of METTL3. To reveal the relationship between the protein structure and function of METTL3, METTL3 were expressed in E. coli, and purified this protein for further study.In this study, dairy cow mettl3 coding sequence was amplified by PCR and was cloned into p GEX-4T-1vector. Protein expression was induced by IPTG and the inclusion bodies was cracked with 8 mol · L-1urea, then GST-METTL3 was purified by protein purified kit, identified by SDS-PAGE and Western blotting analysis. SDS-PAGE confirmed that fusion protein Gst-METTL3 existed in inclusion bodies.Western blotting and SDS-PAGE confirmed the recombinant protein GST-METTL3 was successfully expressed and purified. In summary, this study provided the experimental materials for the further researches on the effect of METTL3 in dairy cow lactation.