MRC Laboratory of Molecular Biology, Hills Road, CB2 0QH Cambridge, United Kingdom.
Bioconjug Chem. 2012 Mar 21;23(3):479-84. doi: 10.1021/bc2005208. Epub 2012 Feb 10.
Combining proteins or their defined domains offers new enhanced functions. Conventionally, two proteins are either fused into a single polypeptide chain by recombinant means or chemically cross-linked. However, these strategies can have drawbacks such as poor expression (recombinant fusions) or aggregation and inactivation (chemical cross-linking), especially in the case of large multifunctional proteins. We developed a new linking method which allows site-oriented, noncovalent, yet irreversible stapling of modified proteins at neutral pH and ambient temperature. This method is based on two distinct polypeptide linkers which self-assemble in the presence of a specific peptide staple allowing on-demand and irreversible combination of protein domains. Here we show that linkers can either be expressed or be chemically conjugated to proteins of interest, depending on the source of the proteins. We also show that the peptide staple can be shortened to 24 amino acids still permitting an irreversible combination of functional proteins. The versatility of this modular technique is demonstrated by stapling a variety of proteins either in solution or to surfaces.
将蛋白质或其定义的结构域组合在一起可以提供新的增强功能。传统上，通过重组手段将两种蛋白质融合成单个多肽链，或者通过化学交联。然而，这些策略可能存在缺点，例如表达不良（重组融合）或聚集和失活（化学交联），特别是对于大型多功能蛋白质。我们开发了一种新的连接方法，允许在中性 pH 和环境温度下对修饰蛋白进行定向、非共价但不可逆的订书钉连接。该方法基于两种不同的多肽接头，它们在存在特定肽钉的情况下自组装，允许按需和不可逆地组合蛋白结构域。在这里，我们表明，接头可以通过表达或化学偶联到感兴趣的蛋白质上，具体取决于蛋白质的来源。我们还表明，肽钉可以缩短至 24 个氨基酸，仍然允许功能性蛋白质的不可逆组合。通过在溶液中或表面上将各种蛋白质进行订书钉连接，证明了这种模块化技术的多功能性。
