不同蛋白标签对LMO2融合蛋白沉淀实验的影响

Effect of Different Tags on Pulldown Assays Implemented by LMO2 Fusion Protein

融合蛋白沉淀技术是一种用来研究蛋白质相互作用的新的体外实验技术,通常利用蛋白亲和标签与探针蛋白融合表达来钓取未知相互作用蛋白或验证已知蛋白间的相互作用,其中以谷胱甘肽巯基转移酶(GST)标签最为常用。LMO2(由LIMonly缩写得名,也称Ttg-2或Rbtn2)是一种小分子量难溶蛋白。利用原核系统分别表达了含有GST和麦芽糖结合蛋白(MBP)两种标签的LMO2融合蛋白,发现GST-LMO2融合蛋白以包涵体的形式表达,而MBP-LMO2融合蛋白则能够以可溶形式表达,而且MBP-LMO2的表达量明显高于GST-LMO2融合蛋白。将可溶性的MBP-LMO2融合蛋白和复性后的GST-LMO2融合蛋白分别用于钓取K562细胞中LMO2的结合蛋白,结果显示二者都可以结合K562细胞中内源性的GATA1蛋白,而MBP-LMO2融合蛋白捕获的GATA1蛋白明显多于复性后的GST-LMO2融合蛋白。这一结果提示,在研究一些分子量小、疏水性强的蛋白质时改变标签蛋白可能是一种有益的尝试。

Pulldown assay is an in vitro method for studies of protein-protein interactions, in which tagged proteins are usually expressed as the bait to enrich other proteins that could bind to them. In this technology, the GST tag is broadest used for its modest size and hydrophilic property. In most cases, the GST tag could increase the hydrophility of the fusion protein and help to avoid the formation of inclusion bodies. However, in the other few cases, the target protein may be strongly hydrophobic or have complicated structures that were hard to fold and assemble in correct conformations without champerons, and even the existence of GST tag could not make them soluble. These proteins were always expressed as inclusion bodies and had no functions. LMO2 was a small molecular weight and insoluble protein, in this study, GST system and MBP system were used to express GST-LMO2 and MBP-LMO2 fusion proteins, respectively. We found that GST-LMO2 fusion protein was expressed as inclusion bodies whereas MBP-LMO2 fusion protein was expressed in soluble form. Moreover, the production rate of MBP-LMO2 was also much higher than GST-LMO2. Then MBP-LMO2 fusion proteins and renatured GST-LMO2 fusion proteins were used as bait in pulldown assay to study the interaction between LMO2 and endogenous GATAI in K562 cells. Western blot analyses showed that both of these proteins could bind to endogenous GATAI in K562 cells, but recovered GATA1 protein by MBP-LMO2 fusion protein was much more than GST-LMO2 fusion protein, These results suggest that using of MBP system is a helpful attempt in the case of studying small molecular weight, strong hydrophobic proteins.