Microscale isolation of native forms of lysozyme from chicken egg white by gel isoelectric focusing
Microscale isolation of native forms of lysozyme from chicken egg white by gel isoelectric focusing
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通过凝胶等电聚焦从鸡蛋清中微量分离天然形式的溶菌酶
DOI：
10.1002/elps.201700445
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发表时间：
2018
期刊：
影响因子：
2.9
通讯作者：
Kennosuke Fujimura
中科院分区：
文献类型：
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作者：
Y. Shimazaki;Yoshiko Ochi;Kennosuke Fujimura
为了从鸡蛋清中分离提取天然溶菌酶，建立了一种非变性凝胶等电聚焦(IEF)结合溶菌酶活性检测的蛋白质动员混合方法。用非变性凝胶离子交换膜首次分离蛋清中的蛋白质时，在离子交换膜阴极端的凝胶柱顶部得到一种溶菌酶。并且，当用磷酸溶液取代阴极氢氧化钠溶液来动员蛋清中IEF分离的蛋白质时，从溶液中提取了可以与蛋白质结合的溶菌酶的额外活性状态，如卵转铁蛋白。此外，通过离子交换获得的溶菌酶加入到纯的卵转铁蛋白中，产生了具有溶菌酶活性的复合体，这清楚地表明溶菌酶-卵转铁蛋白复合体在体外成功地重建了。结果表明，用非变性凝胶IEF可以有效地分离提取溶菌酶和溶菌酶-卵转铁蛋白复合体等溶菌酶的天然状态。因此，该方法可以用于分离和提取保留其生物活性的天然蛋白质的不同电荷状态。
To separate and extract the native states of lysozyme from chicken egg white, a hybrid method for the mobilization of proteins after non‐denaturing gel isoelectric focusing (IEF) combined with detection of lysozyme activity was developed. When the proteins in the chicken egg white were first separated using non‐denaturing gel IEF, a lysozyme was obtained at the top of the gel column at the cathode end of the IEF. And, when the IEF‐separated proteins of the egg white were mobilized by replacing the cathodic sodium hydroxide solution with phosphoric acid solution, an additional active state of the lysozyme that could be bound to proteins, such as ovotransferrin, was extracted from the solution. Furthermore, it was shown that the addition of lysozyme, obtained via IEF, to pure ovotransferrin generated a complex manifesting lysozyme activity, clearly indicating a successful reconstruction of the lysozyme‐ovotransferrin complex in vitro. Therefore, the obtained results demonstrated that the native states of lysozymes, such as lysozyme and the lysozyme‐ovotransferrin complex, can be effectively separated and extracted using non‐denaturing gel IEF. Thus, this method can be applied to separate and extract different charge states of native proteins that retain their biological activities.