牦牛酥油乳脂肪球膜蛋白分离纯化和蛋白组鉴定及功能分析

Isolation,purification,proteome identification and functionalanalysis of yak butter milk fat globular membrane protein

为探究牦牛酥油脂肪球膜(milk fat globule membrane,MFGM)蛋白纯化前后的组成及功能差异,通过表面活性剂聚乙二醇辛基苯基醚溶液和钠离子磷酸盐缓冲溶液提取牦牛酥油中MFGM蛋白,利用AKTA pure 25M蛋白质纯化系统结合阴离子交换层析柱DEAE-Sepharose FF(DEAE琼脂糖凝胶),对MFGM蛋白脂肪酸结合蛋白3(fatty acid binding protein 3,FABP 3)纯化工艺进行优化,得出最优条件:洗脱体积50 mL,缓冲液pH 7.4,洗脱速度1.6 mL/min,洗脱液浓度0.50 mol/L,该条件下,FABP 3得率达83.19%。以牦牛酥油MFGM蛋白粗提物和纯化物为研究对象,通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳和液相色谱质谱联用技术,结合无标记相对定量分析,得出主要MFGM蛋白为FABP 3,共定量到31种MFGM蛋白,其中纯化MFGM蛋白和粗提MFGM蛋白差异蛋白20种,并通过基因本体功能注释、KEGG代谢通路分析对鉴定到的蛋白进行功能及作用的生物学途径探讨。

In order to explore the protein composition and functional differences of milk fat globule membrane(MFGM)proteins,the MFGM proteins in yak butter were extracted with the surfactant polyethylene glycol octyl phenyl ether solution and sodium ion phosphate buffer solution.The purification process of MFGM proteins fatty acid binding protein 3(FABP 3)was optimized by DEAE-Sepharose FF(DEAE agarose gel)using AKTA pure 25M protein purification system.The optimum conditions were as follows:elution volume was 50 mL and buffer pH was 7.4 with the eluent concentration of 0.50 mol/L;elution rate was 1.6 mL/min.Under above conditions,the yield of FABP 3 was 83.19%.The main MFGM protein was FABP 3,and a total of 31 MFGM proteins were identified with the method of SDS-PAGE and liquid chromatography-mass spectrometry combined with unlabeled relative quantitative analysis.There were 20 MFGM differential proteins could be purified and crude extracted.The biological pathways of function and action of the identified proteins were discussed by gene body functional annotation and KEGG metabolic pathway analysis.