Bioconjug Chem. 2018 May 16;29(5):1714-1719. doi: 10.1021/acs.bioconjchem.8b00163. Epub 2018 Apr 30.
Polyproteins, individual protein units joined covalently in tandem, have evolved as a promising tool for measuring the dynamic folding of biomacromolecules in single-molecule force spectroscopy. However, the synthetic routes to prepare polyproteins have been a bottleneck, and urge development of in vitro methods to knit individual protein units covalently into polyprotein. Employing two enzymes of orthogonal functionalities periodically in sequence, we synthesized monodispersed polyproteins on a solid surface. We used Sortase A (SrtA), the enzyme known for sequence specific transpeptidation, to staple protein units covalently through peptide bonds. Exploiting the sequence-specific peptide cleaving ability of TEV protease, we controlled the progress of the reaction to one attachment at a time. Finally, with unique design of the unit proteins we control the orientation of proteins in polyprotein. This simple conjugation has the potential to staple proteins with different functionalities and from different expression systems, in any number in the polyprotein and, above all, via irreversible peptide bonds. Multiple chimeric constructs can also be synthesized with interchangeable protein units.
多聚蛋白是通过共价键串联在一起的单个蛋白质单元，已成为在单分子力谱中测量生物大分子动态折叠的一种很有前途的工具。然而，多聚蛋白的合成途径一直是一个瓶颈，因此迫切需要开发体外方法将单个蛋白质单元共价连接成多聚蛋白。我们采用两种具有正交功能的酶周期性地在序列中依次使用，在固体表面上合成了单分散的多聚蛋白。我们使用已知具有序列特异性转肽作用的 Sortase A（SrtA）通过肽键将蛋白质单元共价连接起来。利用 TEV 蛋白酶的序列特异性肽裂解能力，我们可以控制反应的进展，每次只进行一次连接。最后，通过对单元蛋白的独特设计，我们可以控制多聚蛋白中蛋白质的方向。这种简单的连接有可能将具有不同功能和不同表达系统的蛋白质以任意数量连接在多聚蛋白中，最重要的是通过不可逆的肽键连接。还可以通过可互换的蛋白质单元合成多种嵌合构建体。
