两种跨膜蛋白携带myc于细胞表面的能力比较

Comparison of Two Transmemembrane Proteins as Fusion Partner for Protein Expression on the Surface of Cell

摘要:

在细胞表面表达具有某种功能的蛋白后, 这些细胞就具备了某些新功能, 可以对其进行功能研究和应用。将功能蛋白连接到某些跨膜蛋白的胞外区是实现目的蛋白表达于细胞表面的手段之一。本研究比较了2种跨膜蛋白A2TM和△LNGFR携带外源蛋白－myc于细胞表面的能力。利用重叠PCR法合成的A2TM和△LNGFR基因片段与载体pEF/ myc/ER和载体LL3.7进行酶切、连接、转化、鉴定, 成功构建了pEF-A2TM、pL-A2TM和pL-LN真核表达载体。利用磷酸钙沉淀法转染293FT细胞, 荧光显微镜下观察EGFP的表达、流式细胞术检测myc在细胞表面的表达情况, Western blotting检测myc目的蛋白的表达。pL-A2TM和pL-LN转染293FT细胞36 h后的荧光强度基本相同, 表明A2TM和△LNGFR在细胞内表达的强度相似。流式细胞术分析显示, △LNGFR和A2TM均可在细胞表面表达, 但A2TM只有在指示蛋白EGFP表达非常高的情况下才能在细胞表面被检测到。Western blotting显示△LNGFR表达量很高, 而A2TM没有达到检测水平。结果表明, 以糖基化形式存在的△LNGFR比没有糖基化的内源A2TM的表达更稳定, A2TM在表达量低时可能易被细胞内的蛋白酶降解, 因而△LNGFR是一种能携带外源蛋白于细胞表面的比较理想的跨膜蛋白。

Abstract:

The expression of a soluble protein on cell surface is often desirable for study of a functional protein, wide application of a protein or investigation of protein-protein interaction. The expression of a soluble protein on the surface of a cell is often achieved by genetically linking a protein to the extra-cellular fragment of a transmembrane partner. In this study, the myc epitope was linked with N terminal of transmembrane proteins either A2TM or △LNGFR amplified by overlapping PCR. The plasmids expressing fusion protein were transfected into 293FT cells and the expression of target proteins was evaluated by fluorescent microscope, flow cytometry and Western blotting. The results of flow cytometry revealed that both A2TM and △LNGFR were expressed on the cell surface, but A2TM could only be detected with high copy number. Western blotting showed that the expression level of △LNGFR was very high and protein was heavily glycosylated, by contrast the expression of A2TM was hardly detected. The results indicate that glycosylated △LNGFR is a good candidate partner for the expression of a soluble protein on the cell surface.