Ligation of a synthetic peptide to the N terminus of a recombinant protein using semisynthetic protein trans-splicing
Ligation of a synthetic peptide to the N terminus of a recombinant protein using semisynthetic protein trans-splicing
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DOI：
10.1002/anie.200600570
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发表时间：
2006-01-01
影响因子：
16.6
通讯作者：
Mootz, Henning D.
中科院分区：
文献类型：
--
作者：
Ludwig, Christina;Pfeiff, Martina;Mootz, Henning D.
我们能够制备具有确定共价结构的化学修饰蛋白质，这对在分子水平上理解蛋白质功能有重要贡献。独特的生物化学，生物物理，甚至基于细胞的研究是通过精确引入，例如，荧光团，生物物理探针，非天然氨基酸（aa）与改变侧链或骨架组成，和翻译后修饰。[1]几种技术已被用于位点特异性地将这样的合成结构单元掺入蛋白质中。[2]可能目前最广泛使用的方法是基于天然化学连接（NCL）方法。[2b]它依赖于两种多肽的化学选择性连接，无论是合成的还是重组的，其中一种带有C-末端α-硫酯，另一种带有N-末端半胱氨酸残基，以得到完全合成的或N-或C-末端修饰的半合成蛋白质（综述见参考文献10）。[3]）。尽管如此，反应条件和所需官能团的性质可能限制NCL的范围并使其实际处理复杂化。因为它是一个双分子反应，所以需要高的反应物浓度。在反应物的制备过程中也会遇到问题。特别地，用α-硫酯制备肽或蛋白质可能在技术上要求很高。蛋白质α-硫酯通过感兴趣的蛋白质与修饰的内含肽的融合构建体的硫解产生。[2e获得半合成蛋白质的化学酶促方法包括反向蛋白水解[2a，h，i，4]，其也需要具有活性酯的肽，以及最近报道的分选酶的使用。[2k]在这里，我们表明，蛋白质的反式剪接可用于制备N-末端修饰的半合成蛋白质。我们的方法既不需要合成肽的α-硫酯，也不需要高反应物浓度。
Our ability to prepare chemically modified proteins with defined covalent structure contributes significantly to the understanding of protein function on the molecular level. Unique biochemical, biophysical, and even cell-based investigations are enabled by the precise introduction of, for example, fluorophores, biophysical probes, unnatural amino acids (aa) with altered side chain or backbone composition, and posttranslational modifications.[1] Several techniques have been used to site-specifically incorporate such synthetic building blocks into proteins.[2] Probably the currently most widely used approach is based on the native chemical ligation (NCL) methodology.[2b] It relies on the chemoselective ligation of two polypeptides, either synthetic or recombinant, one of which bears a C-terminal α-thioester and the other anN-terminal cysteine residue, to give a fully synthetic or an N-or C-terminally modified semisynthetic protein (for reviews see Ref.[3]). Nonetheless, the reaction conditions and the nature of the required functional groups can limit the scope and complicate the practical handling of NCL. Because it is a bimolecular reaction, high reactant concentrations are required. Problems can also be encountered during the preparation of the reactants. In particular, the preparation of peptides or proteins with an α-thioester can be technically demanding. Protein α-thioesters are generated by thiolysis of a fusion construct of the protein of interest with a modified intein.[2e, f] Chemoenzymatic approaches to obtain semisynthetic proteins include reverse proteolysis,[2a, h, i, 4] which also requires a peptide with an active ester, and the recently reported use of sortase.[2k] Herein, we show that protein transsplicing can be exploited for the preparation of N-terminally modified semisynthetic proteins. Our approach requires neither an α-thioester at the synthetic peptide nor high reactant concentrations.