非受体型酪氨酸激酶-Syk蛋白的提取与纯化

Extraction and purification of non-receptor type PTKs-Syk

目的 :从转染syk基因的Sf2 1细胞中提取、纯化免疫相关因子Syk蛋白。方法 :将syk基因转染Sf2 1细胞 ,于 2 8℃培养 48h ,收集细胞 ,用超声波破碎仪裂解细胞 ,提取裂解液中总蛋白 ,用Yellow 3凝胶和Toyopearl AF Heptin 650M凝胶层析柱分离、纯化。层析液中的Syk蛋白存在和性质 ,用SDS PAGE、免疫印迹实验和等电聚焦实验鉴定。结果 :从 2 5亿个Sf2 1细胞裂解液中提取了含有Syk的 2 2 5mg蛋白质。经Yellow 3凝胶层析分离 ,得到两个亚种的Syk蛋白 ,相对分子质量 (Mr)均为72× 10 3 。进一步用Toyopearl AF Heptin 650M凝胶层析纯化后 ,得到两个纯的Syk蛋白 ,SDS PAGE、免疫印迹实验结果显示 ,两种Syk的Mr 均为 72× 10 3,与Syk的理论相对分子质量吻合。但等电聚焦实验显示 ,这两种Syk蛋白成分具有不同的pI值。结论 :从 2 5亿个转染syk基因的Sf2 1细胞中纯化出8mgSyk蛋白 ,纯度高于 95%。这两种Syk的Mr 虽然相同 ,但具有不同的pI值 ,是两个亚种。这些Syk可用于研究Syk的作用机制。

AIM: To extract and purify Syk protein from Sf21 cells transfected by syk gene. METHODS: Sf21 cells were transfected with recombinan t syk gene. After 48h of incubation at 28℃, the transfected cells were coll ected and sonicated with Sonfier sonicator on ice. Filtered cell extract was loa ded onto a Reactive Yellow-3 resin column and Toyopearl AF-Heparin-650M colum n respectively. The character of Syk protein in the fractions were identified by SDS-PAGE, Western blotting and IEF. RESULTS: 225 mg of protein containing Syk were obtained from Sf21 cells (2.5×10 9) extract. There were t wo subpopulations in the elution of Reactive Yellow-3 resin column with the sam e relative molecular mass (M r) 72×10 3. The two subpopulations were then applied on Toyopearl AF-Heparin-650M column and two pure proteins were obtain ed. The results of SDS-PAGE, Western blotting, and IEF showed the two proteins having the same relative molecular mass (72×10 3), corresponding to Syk, but w ith different pI. CONCLUSION: The yield of Syk was 8 mg from 2.5 billion cells and the purity was >95%. The two purified Syk proteins have the s ame M r and different pI. The purified Syk protein can be applied to study Syk’s mechanism, produce anti-Syk antibody and invent Sy k diagnosis kit, etc.