Institute of Biotechnology, University of Helsinki, Helsinki, Finland.
FEBS Lett. 2020 Oct;594(20):3338-3355. doi: 10.1002/1873-3468.13909. Epub 2020 Sep 26.
Inteins catalyze self-excision from host precursor proteins while concomitantly ligating the flanking substrates (exteins) with a peptide bond. Noncatalytic extein residues near the splice junctions, such as the residues at the -1 and +2 positions, often strongly influence the protein-splicing efficiency. The substrate specificities of inteins have not been studied for many inteins. We developed a convenient mutagenesis platform termed "QuickDrop"-cassette mutagenesis for investigating the influences of 20 amino acid types at the -1 and +2 positions of different inteins. We elucidated 17 different profiles of the 20 amino acid dependencies across different inteins. The substrate specificities will accelerate our understanding of the structure-function relationship at the splicing junctions for broader applications of inteins in biotechnology and molecular biosciences.
内肽酶在催化自身从宿主前体蛋白中切除的同时，还将侧翼的底物（外肽酶）通过肽键连接起来。在剪接连接处附近的非催化外肽酶残基，例如 -1 和 +2 位的残基，通常会强烈影响蛋白质剪接效率。许多内肽酶的底物特异性尚未得到研究。我们开发了一种名为“QuickDrop”-盒式突变的便利突变平台，用于研究不同内肽酶的 -1 和 +2 位置的 20 种氨基酸类型的影响。我们阐明了不同内肽酶的 20 种氨基酸依赖性的 17 种不同模式。这些底物特异性将加速我们对剪接连接处结构-功能关系的理解，从而更广泛地将内肽酶应用于生物技术和分子生物科学。
