Highly Efficient and More General cis- and trans-Splicing Inteins through Sequential Directed Evolution
Highly Efficient and More General cis- and trans-Splicing Inteins through Sequential Directed Evolution
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DOI：
10.1074/jbc.m111.277350
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发表时间：
2011-09-30
影响因子：
4.8
通讯作者：
Liu, Xiang-Qin
中科院分区：
文献类型：
--
作者：
Appleby-Tagoe, Julia H.;Thiel, Ilka V.;Liu, Xiang-Qin
内含肽是内部蛋白质序列，可在翻译后自我切除并将侧翼序列拼接在一起，即所谓的外显肽。天然和工程内含子已用于许多实际应用。然而，当置于非天然宿主蛋白中时，内含肽通常效率低下或无活性，并且可能需要天然外显肽的几个氨基酸残基的存在，然后这些残基将作为剪接蛋白中的潜在疤痕保留。因此，非常需要克服这些限制的更通用的内含子。在这里，我们报告顺序定向进化作为产生具有此类特性的内含肽的新方法。将 Ssp（集胞藻属 PCC6803）DnaB 迷你内含肽的随机突变体插入到赋予卡那霉素抗性的蛋白质中，该位点位于亲本内含肽无剪接活性的位点。通过在同一蛋白质的不同位置重复该过程两个以上循环，进一步改进了选择用于剪接活性的突变体。由此产生的改进的内含肽在第一轮选择的位置、多个新插入位点以及不同的蛋白质中表现出高活性。这些内含肽之一，M86突变体，积累了8个氨基酸取代，也以人工分裂的形式进行生化表征，其中化学合成的N端内含肽片段由11个氨基酸组成。与未进化的分裂内含肽相比，它在蛋白质转拼反应中表现出类似60倍的增加率，并且分裂内含肽片段相互作用的K-d值提高了一个数量级。讨论了对内含肽结构功能、实际应用和进化的影响。
Inteins are internal protein sequences that post-translationally self-excise and splice together the flanking sequences, the so-called exteins. Natural and engineered inteins have been used in many practical applications. However, inteins are often inefficient or inactive when placed in a non-native host protein and may require the presence of several amino acid residues of the native exteins, which will then remain as a potential scar in the spliced protein. Thus, more general inteins that overcome these limitations are highly desirable. Here we report sequential directed evolution as a new approach to produce inteins with such properties. Random mutants of the Ssp (Synechocystis sp. PCC6803) DnaB mini-intein were inserted into the protein conferring kanamycin resistance at a site where the parent intein was inactive for splicing. The mutants selected for splicing activity were further improved by iterating the procedure for two more cycles at different positions in the same protein. The resulting improved inteins showed high activity in the positions of the first rounds of selection, in multiple new insertion sites, and in different proteins. One of these inteins, the M86 mutant, which accumulated 8 amino acid substitutions, was also biochemically characterized in an artificially split form with a chemically synthesized N-terminal intein fragment consisting of 11 amino acids. When compared with the unevolved split intein, it exhibited an similar to 60-fold increased rate in the protein trans-splicing reaction and a K-d value for the interaction of the split intein fragments improved by an order of magnitude. Implications on the intein structure-function, practical application, and evolution are discussed.