植物和微生物凝集素的提取纯化及特性比较分析
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摘要
采用Sepharose CL-6B亲和层析提取分离三种植物种子(白果、苦瓜籽、蓖麻子)和大肠杆菌(Escherichia coli, E.coli)中的半乳糖结合凝集素。凝血实验以及还原性和非还原性SDS-PAGE(十二烷基磺酸钠-聚丙烯酰胺凝胶电泳)检测所提取蛋白的凝血特性和分子构成；并对所提取的4种不同蛋白进行比较,分析其在进化上的关系。
实验结果显示,目标蛋白经过简单的步骤即可得到纯化。提取所得的4种不同的目的蛋白均具有凝血效果,且半乳糖均可以抑制这4种蛋白的凝集作用,而葡萄糖对其却没有抑制效果,证明实验所提取的目的蛋白为半乳糖结合凝集素。还原性和非还原性SDS-PAGE实验结果显示,白果凝集素(SGA)是单亚基蛋白,分子量约为32kDa；蓖麻凝集素(RCA1)和苦瓜凝集素(MCL)均为4亚基蛋白,分子量分别约为120kDa和116kDa。而E. coli半乳糖结合凝集素(EGA)也是单亚基蛋白,但其分子量约为45kDa。对研究结果的进一步分析表明,SGA只有一条结合半乳糖的B链亚基(糖结合位点识别链,约32kDa),而RCA1和MCL则是在B链亚基的基础上演化而成的四聚体蛋白。未发现EGA与其他三种植物半乳糖结合凝集素的同源关系。
研究证明,Sepharose CL-6B亲和层析在提取分离半乳糖结合凝集素,尤其是含有B链亚基的凝集素的应用上有巨大的优势和潜力。本研究不仅为植物和微生物中的半乳糖结合凝集素的纯化提供了理想的方法,而且对认识半乳糖结合凝集素在进化史上的演化提供了初步的理论支持。
Sepharose CL-6B affinity chromatography was adopt in this study to separate the galactose-binding lectins from three different plants seeds of Ginkgo biloba L.(Semen Ginkgo), Momordica charantial and Ricinus communi, and Escherichia coli (E.coli). Hemagglutination experiment showed that four target proteins possessed the characteristic of hemagglutination. Addition of galactose inhibited hemagglutination of these proteins, while glucose had no effect on it. This indicated that the target proteins were galactose-binding proteins. Reducing and Non-reducing Sodium dodecyl sulfate Polyacrylamide gel electrophoresis (SDS-PAGE) was taken to detect the molecular weight and subunits constitution of target proteins. B-chain subunit (recognition chain to sugar-binding site,32kDa) was found in all three proteins of Semen ginkgo agglutinin (SGA), Ricinus communis agglutinin (RCA1) and Momordica charantial lectin (MCL). SGA was a single protein, while MCL and RCA1 were tetrameric proteins, with molecular weight 32kDa,116kDa and 120kDa, respectively. Also E.coli galatose-binding lectin (EGA) was a single protein, with molecular weight 45kDa, but no homology relations was found between EGA and all three target plant lectins in this study.
Sepharose CL-6B affinity chromatography showed its advantages and potential in purification of galactose-binding lectins, especially lectins with B-chain subunit. This research provides a novel and rapid method for the purification of galactose-binding lectins from plant and microbial, and may offer the initial theoretical support to the evolution history of galactose-binding lectins.
实验结果显示,目标蛋白经过简单的步骤即可得到纯化。提取所得的4种不同的目的蛋白均具有凝血效果,且半乳糖均可以抑制这4种蛋白的凝集作用,而葡萄糖对其却没有抑制效果,证明实验所提取的目的蛋白为半乳糖结合凝集素。还原性和非还原性SDS-PAGE实验结果显示,白果凝集素(SGA)是单亚基蛋白,分子量约为32kDa；蓖麻凝集素(RCA1)和苦瓜凝集素(MCL)均为4亚基蛋白,分子量分别约为120kDa和116kDa。而E. coli半乳糖结合凝集素(EGA)也是单亚基蛋白,但其分子量约为45kDa。对研究结果的进一步分析表明,SGA只有一条结合半乳糖的B链亚基(糖结合位点识别链,约32kDa),而RCA1和MCL则是在B链亚基的基础上演化而成的四聚体蛋白。未发现EGA与其他三种植物半乳糖结合凝集素的同源关系。
研究证明,Sepharose CL-6B亲和层析在提取分离半乳糖结合凝集素,尤其是含有B链亚基的凝集素的应用上有巨大的优势和潜力。本研究不仅为植物和微生物中的半乳糖结合凝集素的纯化提供了理想的方法,而且对认识半乳糖结合凝集素在进化史上的演化提供了初步的理论支持。
Sepharose CL-6B affinity chromatography was adopt in this study to separate the galactose-binding lectins from three different plants seeds of Ginkgo biloba L.(Semen Ginkgo), Momordica charantial and Ricinus communi, and Escherichia coli (E.coli). Hemagglutination experiment showed that four target proteins possessed the characteristic of hemagglutination. Addition of galactose inhibited hemagglutination of these proteins, while glucose had no effect on it. This indicated that the target proteins were galactose-binding proteins. Reducing and Non-reducing Sodium dodecyl sulfate Polyacrylamide gel electrophoresis (SDS-PAGE) was taken to detect the molecular weight and subunits constitution of target proteins. B-chain subunit (recognition chain to sugar-binding site,32kDa) was found in all three proteins of Semen ginkgo agglutinin (SGA), Ricinus communis agglutinin (RCA1) and Momordica charantial lectin (MCL). SGA was a single protein, while MCL and RCA1 were tetrameric proteins, with molecular weight 32kDa,116kDa and 120kDa, respectively. Also E.coli galatose-binding lectin (EGA) was a single protein, with molecular weight 45kDa, but no homology relations was found between EGA and all three target plant lectins in this study.
Sepharose CL-6B affinity chromatography showed its advantages and potential in purification of galactose-binding lectins, especially lectins with B-chain subunit. This research provides a novel and rapid method for the purification of galactose-binding lectins from plant and microbial, and may offer the initial theoretical support to the evolution history of galactose-binding lectins.
引文
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