Reprogramming the specificity of sortase enzymes
Reprogramming the specificity of sortase enzymes
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DOI：
10.1073/pnas.1411179111
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发表时间：
2014-09-16
影响因子：
11.1
通讯作者：
Liu, David R.
中科院分区：
文献类型：
--
作者：
Dorr, Brent M.;Ham, Hyun Ok;Liu, David R.
金黄色葡萄球菌分类酶A催化LPXTG多肽受体和甘氨酸连接多肽供体的转肽化，已被证明是一种强有力的位点特异性蛋白质修饰工具。分类酶A的底物专一性很强，限制了其更广泛的用途。在这里，我们报告了两种可识别两种改变的底物LAXTG和LPXSG的两种正交索酸酶A变体的实验室进化，具有高活性和特异性。经过9轮酵母展示筛选和阴性选择相结合，进化的分类酶相对于起始分类酶表现出高达51,000倍的特异性变化，而催化活性没有实质性的损失；相对于它们的下一个最活跃的多肽底物，它们对目标底物的特异性高达24倍。这些改变的分解酶的特性是足够的正交性，能够在单个溶液中同时将多个多肽底物与其各自的靶标结合。我们通过使用这些进化的索酸酶对人血浆中内源性胎球蛋白A进行位置特异性修饰，合成两种具有治疗意义的成纤维细胞生长因子蛋白(FGF1和FGF2)的串联荧光团-蛋白质-聚乙二醇偶联物，以及荧光肽在表面的正交偶联物，展示了这些进化的索糖酶的实用性。
Staphylococcus aureus sortase A catalyzes the transpeptidation of an LPXTG peptide acceptor and a glycine-linked peptide donor and has proven to be a powerful tool for site-specific protein modification. The substrate specificity of sortase A is stringent, limiting its broader utility. Here we report the laboratory evolution of two orthogonal sortase A variants that recognize each of two altered substrates, LAXTG and LPXSG, with high activity and specificity. Following nine rounds of yeast display screening integrated with negative selection, the evolved sortases exhibit specificity changes of up to 51,000-fold, relative to the starting sortase without substantial loss of catalytic activity, and with up to 24-fold specificity for their target substrates, relative to their next most active peptide substrate. The specificities of these altered sortases are sufficiently orthogonal to enable the simultaneous conjugation of multiple peptide substrates to their respective targets in a single solution. We demonstrated the utility of these evolved sortases by using them to effect the site-specific modification of endogenous fetuin A in human plasma, the synthesis of tandem fluorophore-protein-PEG conjugates for two therapeutically relevant fibroblast growth factor proteins (FGF1 and FGF2), and the orthogonal conjugation of fluorescent peptides onto surfaces.