A chemoenzymatic approach enables the site-specific conjugation of recombinant proteins
A chemoenzymatic approach enables the site-specific conjugation of recombinant proteins
复制标题
化学酶方法能够实现重组蛋白的位点特异性缀合
DOI：
10.1002/elps.201800528
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发表时间：
2019
期刊：
影响因子：
2.9
通讯作者：
Ye Mingliang
中科院分区：
文献类型：
--
作者：
Wang Yan;Dong Mingming;Ye Mingliang
许多生物技术，包括蛋白质微阵列、药物筛选、生物传感器，都依赖于重组蛋白质在固相支持物上的固定。众所周知，固定蛋白的随机方向可能会损害其生物功能。因此，开发新的位点特异性固定方法非常重要。在这项研究中，我们提出了一种将重组蛋白位点特异性缀合到固体支持物上的化学酶方法。在该策略中，重组蛋白上的亲和标签被酶促切割以暴露N末端丝氨酸，丝氨酸被氧化以携带醛基，然后通过腙连接与酰肼树脂共价偶联。由于这种方法利用了最常用的 TEV 蛋白酶，因此不需要对重组蛋白进行进一步的序列设计。该方法通过合成肽和重组蛋白在固体支持物上的位点特异性偶联进行了验证。结果发现，位点特异性固定的 SH2 结构域具有功能性，可用于富集酪氨酸磷酸化肽。
Many biotechniques including protein microarray, drug screening, biosensors rely on the immobilization of recombinant proteins on the solid supports. It is well known that random orientation of the immobilized proteins could impair their biologic functions. Thus, it is very important to develop new site‐specific immobilization approach. In this study, we presented a chemoenzymatic approach for site‐specific conjugation of recombinant proteins onto solid support. In this strategy, the affinity tag on recombinant protein was enzymatically cleaved to expose the N‐terminal serine, which was oxidized to carry an aldehyde group and was then covalently coupled to hydrazide resin through hydrazone ligation. As this approach takes advantage of the most frequently used TEV protease, it requires no further sequence design on recombinant protein. This method was validated by site specific coupling of a synthetic peptide and a recombinant protein onto solid supports. It was found that the site specific immobilized SH2 domain is functional and could be used to enrich tyrosine phosphorylated peptides.