SELECTIVE ENZYME PURIFICATION BY AFFINITY CHROMATOGRAPHY
SELECTIVE ENZYME PURIFICATION BY AFFINITY CHROMATOGRAPHY
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DOI：
10.1073/pnas.61.2.636
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发表时间：
1968-01-01
影响因子：
11.1
通讯作者：
ANFINSEN, CB
中科院分区：
文献类型：
--
作者：
CUATRECA.P;WILCHEK, M;ANFINSEN, CB
通过常规方法纯化蛋白质通常是费力和不完全的，并且产率通常很低。基于高度特异性的生物学性质--与特定底物或底物的强可逆缔合--的酶分离只受到有限的关注。1-4在亲和层析中，待纯化的酶通过含有交联聚合物或凝胶的柱，酶的特异性竞争性抑制剂已共价连接到所述交联聚合物或凝胶上。所有对结合的抑制剂没有实质性亲和力的蛋白质将直接通过柱，而识别抑制剂的蛋白质将与其亲和力常数成比例地被阻滞。结合酶的洗脱很容易通过改变这些参数如盐作为盐浓度或pH，或通过在溶液中加入竞争性抑制剂来实现。因此，未取代的基质或凝胶在与特异性结合基团偶联之前和之后通常应显示与蛋白质的最小相互作用。它必须形成一个松散的多孔网络，允许大分子容易进入和离开，并在使用过程中保持良好的流动性能。支持材料的化学结构必须允许特异性配体在相对温和的条件下，通过对吸附和洗脱条件稳定的化学键方便和广泛地连接。最后，在相互作用中起关键作用的抑制剂基团必须离固体基质足够远，以最大限度地减少对结合过程的空间干扰。本文通过亲和层析在葡萄球菌核酸酶、α-糜蛋白酶和羧肽酶A纯化中的应用结果，阐明了亲和层析的一般原理和潜在应用。在这些研究中使用的固体基质是琼脂糖凝胶（高度多孔结构的交联葡聚糖的”珠状”形式，琼脂糖5），其显示出几乎所有上述所需的特征。通过用溴化氰处理活化琼脂糖凝胶6 7，产生可以容易地偶联至未取代的氨基allg的衍生物。所得的琼脂糖抑制剂凝胶是一种高度稳定的结构，具有几乎理想的选择性柱色谱性能。
The purification of proteins by conventional procedures is frequently laborious and incomplete, and the yields are often low. Enzyme isolation based on a highly specific biological property-strong reversible association with specific substrates or inhibitors-has received only limited attention. 1-4 In affinity chromatography, the enzyme to be purified is passed through a column containing a cross-linked polymer or gel to which a specific competitive inhibitor of the enzyme has been covalently attached. All proteins without substantial affinity for the bound inhibitor will pass directly through the column, whereas one that recognizes the inhibitor will be retarded in proportion to its affinity constant. Elution of the bound enzyme is readily achieved by changing such parameters as salt concentration or pH, or by addition of a competitive inhibitor in solution.The successful application of the method requires that the adsorbent have a number of favorable characteristics. Thus, the unsubstituted matrix or gel should show minimal interaction with proteins in general, both before and after coupling to the specific binding group. It must form a loose, porous network that permits easy entry and exit of macromolecules and which retains favorable flow properties during use. The chemical structure of the supporting material must permit the convenient and extensive attachment of the specific ligand under relatively mild conditions, and through chemical bonds that are stable to the conditions of adsorption and elution. Finally, the inhibitor groups critical in the interaction must be sufficiently distant from the solid matrixto minimize steric interference with the binding processes. In this report the general principles and potential application of affinity chro-matography are illustrated byresults of its application to the purification of staphylococcal nuclease, a-chymotrypsin, and carboxypeptidase A. The solid matrix used in these studies was Sepharose (a" beaded" form of the cross-linked dextran of highly porous structure, agarose5) which displays virtually all the desirable fea-tures listed above. Activation of the Sepharose by treatment with cyanogen bromide6 7 results in aderivative that can be readily coupled to unlrotonated amino allg. The resultant Sepharose-inhibitor gel is a highly stablestructure which has nearly ideal properties for selectivecolumn chromatography.