Site-specific PEGylation of a lysine-deficient TNF-α with full bioactivity
Site-specific PEGylation of a lysine-deficient TNF-α with full bioactivity
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DOI：
10.1038/nbt812
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发表时间：
2003-05
影响因子：
46.9
通讯作者：
Yoko Yamamoto;Y. Tsutsumi;Y. Yoshioka;T. Nishibata;Kyoko Kobayashi;T. Okamoto;Y. Mukai;Tomoe Shimizu;S. Nakagawa;S. Nagata;T. Mayumi
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作者：
Yoko Yamamoto;Y. Tsutsumi;Y. Yoshioka;T. Nishibata;Kyoko Kobayashi;T. Okamoto;Y. Mukai;Tomoe Shimizu;S. Nakagawa;S. Nagata;T. Mayumi
向蛋白质中添加聚乙二醇（PEG化）以改善稳定性和其他特性大多是非特异性的，并且可能发生在所有赖氨酸残基上，其中一些可能在活性位点内或附近。所得聚乙二醇化蛋白质是异质的，并且可显示出显著较低的生物活性。我们试图开发一种使用肿瘤坏死因子-α（TNF-α）进行位点特异性单聚乙二醇化的策略。我们制备了表达TNF-α突变体的噬菌体文库，其中所有的赖氨酸残基都被其他氨基酸取代。尽管有报道称某些赖氨酸残基是其生物活性所必需的，但通过对TNF-α中和抗体的淘选，分离出了一种完全具有生物活性的赖氨酸缺陷型突变TNF-α（mTNF-α-Lys（−））。mTNF-α-Lys（−）在其N末端被位点特异性单聚乙二醇化。这种单聚乙二醇化的mTNF-α-Lys（-）具有上级分子均匀性，与随机单聚乙二醇化的野生型TNF-α相比，显示出更高的体外生物活性和更大的抗肿瘤治疗效力。这些结果表明噬菌体展示系统用于产生功能突变蛋白和我们的位点特异性PEG化方法的有用性。
Addition of polyethylene glycol to protein (PEGylation) to improve stability and other characteristics is mostly nonspecific and may occur at all lysine residues, some of which may be within or near an active site. Resultant PEGylated proteins are heterogeneous and can show markedly lower bioactivity. We attempted to develop a strategy for site-specific mono-PEGylation using tumor necrosis factor-α (TNF-α). We prepared phage libraries expressing TNF-α mutants in which all the lysine residues were replaced with other amino acids. A fully bioactive lysine-deficient mutant TNF-α (mTNF-α-Lys(−)) was isolated by panning against TNF-α-neutralizing antibody despite reports that some lysine residues were essential for its bioactivity. mTNF-α-Lys(−) was site-specifically mono-PEGylated at its N terminus. This mono-PEGylated mTNF-α-Lys(−), with superior molecular uniformity, showed higher bioactivityin vitroand greater antitumor therapeutic potency than randomly mono-PEGylated wild-type TNF-α. These results suggest the usefulness of the phage display system for creating functional mutant proteins and of our site-specific PEGylation approach.