Recent progress in intein research: from mechanism to directed evolution and applications

Abstract

Inteins catalyze a post-translational modification known as protein splicing, where the intein removes itself from a precursor protein and concomitantly ligates the flanking protein sequences with a peptide bond. Over the past two decades, inteins have risen from a peculiarity to a rich source of applications in biotechnology, biomedicine, and protein chemistry. In this review, we focus on developments of intein-related research spanning the last 5 years, including the three different splicing mechanisms and their molecular underpinnings, the directed evolution of inteins towards improved splicing in exogenous protein contexts, as well as novel applications of inteins for cell biology and protein engineering, which were made possible by a clearer understanding of the protein splicing mechanism.

Cyclin-dependent kinase 4 may be expressed as multiple proteins and have functions that are independent of binding to CCND and RB and occur at the S and G2/M phases of the cell cycle

“…35,36 Many types of post-translational modifications, including less discussed S-nitrosylation 37 and many non-enzymatic reactions 38 can also affect protein mobility in SDS-PAGE. Indeed, so often western blots detect multiple bands, although most publications only present the anticipated band without really proving that the unexpected bands are, in fact, artifacts.…”

pH-Dependent Activation of Streptomyces hygroscopicus Transglutaminase Mediated by Intein

“…Inteins were discovered 2 decades ago, and they have already become a powerful tool in protein engineering (12). The SDB used in this study is one of the inteins used in the IMPACT system (New England BioLabs Inc.), which has widespread applications in protein expression and purification (15), ligation (40), immobilization (16), and cyclization (17).…”

“…Protein splicing is an extraordinary posttranslational processing event in which an internal polypeptide (intein) is excised from a protein precursor, and then the N-terminal polypeptide (Nextein) and C-terminal polypeptide (C-extein) are ligated together (12). Inteins can catalyze protein splicing without the requirement for cofactors or auxiliary enzymes (12).…”

“…Inteins can catalyze protein splicing without the requirement for cofactors or auxiliary enzymes (12). Furthermore, protein splicing can be modulated by mutation and converted to highly specific self-cleavage and protein ligation reactions that are useful protein engineering tools (12).…”

“…Furthermore, protein splicing can be modulated by mutation and converted to highly specific self-cleavage and protein ligation reactions that are useful protein engineering tools (12). Inteins have progressed from being a peculiarity to being widely applied in biotechnology, biomedicine, and protein chemistry (12). A sequence for an intein with 429 amino acids was found in the dnaB gene, encoding a DNA helicase in the cyanobacterium Synechocystis sp.…”