蛋白质剪接是一种自催化过程,其中内部蛋白质结构域(内含肽)从其侧翼序列中切除,用典型的肽键将它们连接在一起。反式内含肽在两个不同的前体多肽链中被分离,所述前体多肽链必须组装以催化自切除并连接相应的侧翼外显子,即使在体外或体内单独表达时也是如此。它们从单独的结构域构建完整的蛋白质是非常有趣的,因为它们共同的小尺寸有利于化学合成方法。因此,反式内含肽具有多种应用,如蛋白质修饰和纯化,蛋白质结构域的结构表征或基于内含肽的生物传感器的生产,在其他人中。对于许多这样的应用,当使用多个trans-intein时,它们之间的正交性是确保外皮正确连接的关键问题。这里,我们确认了四个不同的反式或分裂内含子的正交性(缺乏交叉反应性),gp41-1,gp41-8,IMPDH-1和NrdJ-1在体内和体外,并构建了不同的构建体,可以将多达四个蛋白质片段顺序融合成一个最终的剪接产物。我们已经表征了这些构建体的剪接效率。所有在反式-内含肽和邻近的外显子之间的剪接连接处都有非天然的外显子残基,除了必要的Ser+1。我们的结果表明,有可能使用内含肽gp41-1,IMPDH-1和NrdJ-1与非天然外显子残基连接四个不同的蛋白质结构域,以获得最终的四结构域剪接产物,其产量不可忽略,保持其天然序列。
Protein splicing is a self-catalyzed process in which an internal protein domain (the intein) is excised from its flanking sequences, linking them together with a canonical peptide bond. Trans-
inteins are separated in two different precursor polypeptide chains that must assemble to catalytically self-excise and ligate the corresponding flanking exteins to join even when expressed separately either in vitro or in vivo. They are very interesting to construct full proteins from separate domains because their common small size favors chemical synthesis approaches. Therefore, trans-
inteins have multiple applications such as protein modification and purification, structural characterization of protein domains or production of intein-based biosensors, among others. For many of these applications, when using more than one trans-intein, orthogonality between them is a critical issue to ensure the proper ligation of the exteins. Here, we confirm the orthogonality (lack of cross-reactivity) of four different trans- or split
inteins, gp41-1, gp41-8, IMPDH-1 and NrdJ-1 both in vivo and in vitro, and built different constructs that allow for the sequential fusion of up to four protein fragments into one final spliced product. We have characterized the splicing efficiency of these constructs. All harbor non-native extein residues at the splice junction between the trans-intein and the neighboring exteins, except for the essential Ser + 1. Our results show that it is possible to ligate four different protein domains using
inteins gp41-1, IMPDH-1 and NrdJ-1 with non-native extein residues to obtain a final four-domain spliced product with a not negligible yield that keeps its native sequence.