Efficient Sortase-Mediated Ligation Using a Common C-Terminal Fusion Tag.
Efficient Sortase-Mediated Ligation Using a Common C-Terminal Fusion Tag.
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DOI：
10.1021/acs.bioconjchem.0c00156
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发表时间：
2020-04
影响因子：
4.7
通讯作者：
Sierra Reed;David Brzovic;S. Takasaki;Kristina V Boyko;John M. Antos
中科院分区：
文献类型：
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作者：
Sierra Reed;David Brzovic;S. Takasaki;Kristina V Boyko;John M. Antos
分选酶介导的连接是用于产生位点特异性修饰的蛋白质的有力方法。然而，该过程受到连接反应固有可逆性的限制。为了解决这个问题，在这里，我们报告的持续发展和优化的实验简便的策略，阻断反应的可逆性。这种方法，我们称之为金属辅助分选酶介导的连接（MA-SML），依赖于使用溶液添加剂（Ni 2+）和C-末端标签（LPXTGGHH 5），广泛用于将蛋白质靶转化为分选酶底物。在利用1：1摩尔比的分选酶底物和甘氨酸胺亲核试剂的一系列模型系统中，我们发现MA-SML一致地提高了连接的程度。这使得能够用荧光团、PEG和生物正交环辛炔部分修饰蛋白质，而不需要过量使用珍贵的试剂。总体而言，这些结果证明了MA-SML作为在广泛的基于分选酶的蛋白质工程应用中提高反应效率的一般策略的潜力。
Sortase-mediated ligation is a powerful method for generating site-specifically modified proteins. However, this process is limited by the inherent reversibility of the ligation reaction. To address this, here we report the continued development and optimization of an experimentally facile strategy for blocking reaction reversibility. This approach, which we have termed metal-assisted sortase-mediated ligation (MA-SML), relies on the use of a solution additive (Ni2+) and a C-terminal tag (LPXTGGHH5) that is widely used for converting protein targets into sortase substrates. In a series of model systems utilizing a 1:1 molar ratio of sortase substrate and glycine amine nucleophile, we find that MA-SML consistently improves the extent of ligation. This enables the modification of proteins with fluorophores, PEG, and a bioorthogonal cyclooctyne moiety without the need to use precious reagents in excess. Overall, these results demonstrate the potential of MA-SML as a general strategy for improving reaction efficiency in a broad range of sortase-based protein engineering applications.