Recombinant Protein Hydrazides: Application to Site-Specific Protein PEGylation
Recombinant Protein Hydrazides: Application to Site-Specific Protein PEGylation
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DOI：
10.1021/bc2001374
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发表时间：
2011-06-01
影响因子：
4.7
通讯作者：
Cotton, Graham
中科院分区：
文献类型：
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作者：
Thom, Jennifer;Anderson, David;Cotton, Graham
在这里，我们描述了一种新的方法，用于重组蛋白的位点特异性的C-末端聚乙二醇化。这种通用方法利用前体内含肽融合蛋白与肼的化学切割来直接产生重组蛋白酰肼。然后，蛋白质序列内的这种独特功能性通过腙形成连接反应促进位点特异性C-末端修饰。该方法用于产生折叠的、位点特异性C-末端PEG化的IFN α 2b和IFN β 1b，其保留了优异的抗病毒活性，证明了该技术在治疗性蛋白质的PEG化中的实用性。由于该方法简单易行，与二硫键相容，并且仅对蛋白质C-末端具有选择性，因此它作为重组蛋白的位点特异性工程化和标记的通用技术显示出巨大的潜力。
Here, we describe a novel method for the site-specific C-terminal PEGylation of recombinant proteins. This general approach exploits chemical cleavage of precursor intein-fusion proteins with hydrazine to directly produce recombinant protein hydrazides. This unique functionality within the protein sequence then facilitates site-specific C-terminal modification by hydrazone-forming ligation reactions. This approach was used to generate folded, site-specifically C-terminal PEGylated IFNalpha2b and IFNbeta1b, which retained excellent antiviral activity, demonstrating the utility of this technology in the PEGylation of therapeutic proteins. As this methodology is straightforward to perform, is compatible with disulfide bonds, and is exclusively selective for the protein C-terminus, it shows great potential as general technology for the site-specific engineering and labeling of recombinant proteins.