Improved variants of SrtA for site-specific conjugation on antibodies and proteins with high efficiency.
Improved variants of SrtA for site-specific conjugation on antibodies and proteins with high efficiency.
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DOI：
10.1038/srep31899
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发表时间：
2016-08-18
影响因子：
4.6
通讯作者：
Chen PR
中科院分区：
文献类型：
--
作者：
Chen L;Cohen J;Song X;Zhao A;Ye Z;Feulner CJ;Doonan P;Somers W;Lin L;Chen PR
索尔特酶介导的连接是一种高度特异的连接平台，它依赖于短肽序列(LPXTG和GGG)的转肽酶A(SrtA)的特异性。SrtA在接受多种潜在底物的同时保持了其特异性，但其广泛应用受到野生型酶动力学较差的限制，这需要大量的srtA和较长的反应时间才能有效地连接。先前的探索旨在改善srtA的动力学，但收效甚微。在这里，我们描述了进一步改进的srtA变异体的发现，提高了接合反应的效率，并展示了它们以特定部位的方式标记蛋白质和抗体的稳健性。我们的变异体需要的酶比WT SrtA低得多，可以用来将小分子连接到抗体的重链或轻链的N端或C端，产量很高。这些改进的变体也可以用于高效的位点特异性聚乙二醇化。
Sortase mediated ligation is a highly specific platform for conjugation that relies on the specificity of the transpeptidase Sortase A (SrtA) for short peptide sequences (LPXTG and GGG). SrtA retains its specificity while accepting a wide range of potential substrates, but its broad use is limited by the wild-type enzyme’s poor kinetics, which require large amounts of SrtA and extended reaction times for efficient conjugation. Prior explorations have aimed to improve the kinetics of SrtA with limited success. Herein we describe the discovery of further improved SrtA variants with increased efficiency for the conjugation reaction, and demonstrate their robustness in labelling proteins and antibodies in a site-specific manner. Our variants require significantly lower amounts of enzyme than WT SrtA and can be used to attach small molecules to the N or C-terminus of the heavy or light chain in antibodies with excellent yields. These improved variants can also be used for highly efficient site-specific PEGylation.