Total chemical synthesis, folding, and assay of a small protein on a water-compatible solid support
Total chemical synthesis, folding, and assay of a small protein on a water-compatible solid support
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DOI：
10.1002/anie.200600381
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发表时间：
2006-01-01
影响因子：
16.6
通讯作者：
Kent, Stephen B. N.
中科院分区：
文献类型：
--
作者：
Johnson, Erik C. B.;Durek, Thomas;Kent, Stephen B. N.
无保护肽的化学连接使大量不同蛋白质的成功合成成为可能天然化学连接（NCL）反应[2]在中性pH的水中进行，在连接位点产生天然酰胺键，已被证明是无保护肽连接最有效的化学反应。NCL合成蛋白质和所有肽化学化方法的一个主要限制是，由于中间处理和纯化的损失，产量不断下降。尽管最近化学蛋白合成的一锅方案有助于消除一些中间纯化步骤，从而提高产量，但随着时间的推移和多次连接，一锅反应可能导致难以从目标多肽中纯化的大量副产物的积累。对于某些种类的蛋白质，如整体膜蛋白，另一个主要问题是许多中间多肽产物在用于结扎的溶剂中的溶解度差。目前没有有效和通用的策略来溶解在连接缓冲液中的难溶性中间多肽，而不引入对肽构建块的修饰。我们的目标是通过NCL在交联聚合物载体上进行蛋白质合成，以解决这些限制。
Chemical ligation of unprotected peptides has enabled the successful synthesis of a large number of diverse proteins.[1] The native chemical ligation (NCL) reaction,[2] which proceeds in water at neutral pH to yield a native amide bond at the ligation site, has proven to be the most effective chemistry for the ligation of unprotected peptides. A major limitation of protein synthesis by NCL, and indeed all peptide chemicalligation methods, is the steady decrease in yield because of losses from intermediate handling and purification. Although recent one-pot schemes [3] for chemical protein synthesis have helped eliminate some intermediate purification steps and, therefore, increased yields, one-pot reactions can, over time and multiple ligations, lead to a large accumulation of byproducts that are difficult to purify from the target polypeptide. For certain classes of proteins, such as integral membrane proteins, an additional major problem is the poor solubility of many intermediate polypeptide products in the solvents used for ligation. There is currently no effective and general strategy for solubilizing poorly soluble intermediate polypeptides in ligation buffers without introducing modifications to the peptide building blocks. We aimed to perform protein synthesis by NCL on a cross-linked polymer support to address these limitations.