SnoopLigase Catalyzes Peptide-Peptide Locking and Enables Solid-Phase Conjugate Isolation
SnoopLigase Catalyzes Peptide-Peptide Locking and Enables Solid-Phase Conjugate Isolation
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DOI：
10.1021/jacs.7b13237
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发表时间：
2018-02-28
影响因子：
15
通讯作者：
Howarth, Mark
中科院分区：
文献类型：
--
作者：
Buldun, Can M.;Jean, Jisoo X.;Howarth, Mark
连接生物构件的简单、有效的反应开辟了许多新的可能性。在这里，我们设计了SnoopLigase，这是一种催化位点特异性转酰胺作用的蛋白质，在两个肽标签SnoopTagJr和DogTag之间以超过95%的效率形成异肽键。我们最初开发这些组件的肺炎链球菌粘附素RrgA的三部分分裂。然后通过结构、序列同源性的生物信息学分析和稳定性的计算预测来工程化这些单元。经过改造，SnoopLigase在宽范围的缓冲液和温度下表现出高产率偶联。SnoopTagJr和DogTag在N-或C-末端起作用，而DogTag也在蛋白质的内部位点起作用。在SnoopTagJr和DogTag的定向反应后，SnoopLigase保持与连接产物稳定结合，从而重建亲本结构域。从未反应的原料和催化剂中分离产物通常与反应本身一样具有挑战性。然而，SnoopLigase的固相固定使得连接的SnoopTagJr-DogTag产物能够以高纯度洗脱，不含SnoopLigase或未连接的底物。固相催化剂可以重复使用多次。为了寻找提高酶弹性的通用途径，我们将SnoopTagJr融合到模型酶的N-末端，将DogTag融合到C-末端，允许通过SnoopLigase进行环化。虽然野生型植酸酶和β-内酰胺酶在加热时不可逆地聚集，但使用SnoopLigase的环化赋予了优异的热弹性，两种酶在高达100摄氏度的热处理后均保持溶解度和活性。SnoopLigase应该为缀合和纳米组装创造新的机会，同时说明如何利用产物抑制和扩展催化剂效用。
Simple, efficient reactions for connecting biological building-blocks open up many new possibilities. Here we have designed SnoopLigase, a protein that catalyzes site-specific transamidation, forming an isopeptide bond with more than 95% efficiency between two peptide tags, SnoopTagJr and DogTag. We initially developed these components by three-part splitting of the Streptococcus pneumoniae adhesin RrgA. The units were then engineered, guided by structure, bioinformatic analysis of sequence homology, and computational prediction of stability. After engineering, SnoopLigase demonstrated high-yield coupling under a wide range of buffers and temperatures. SnoopTagJr and DogTag were functional at the N- or C-terminus, while DogTag was also functional at internal sites in proteins. Having directed reaction of SnoopTagJr and DogTag, SnoopLigase remained stably bound to the ligated product, thus reconstituting the parent domain. Separating products from unreacted starting material and catalyst is often as challenging as reactions themselves. However, solid-phase immobilization of SnoopLigase enabled the ligated SnoopTagJr-DogTag product to be eluted with high purity, free from SnoopLigase or unligated substrates. The solid-phase catalyst could then be reused multiple times. In search of a generic route to improve the resilience of enzymes, we fused SnoopTagJr to the N-terminus and DogTag to the C-terminus of model enzymes, allowing cyclization via SnoopLigase. While wild-type phytase and beta-lactamase irreversibly aggregated upon heating, cyclization using SnoopLigase conferred exceptional thermoresilience, with both enzymes retaining solubility and activity following heat treatment up to 100 degrees C. SnoopLigase should create new opportunities for conjugation and nanoassembly, while illustrating how to harness product inhibition and extend catalyst utility.