Ultrafast Protein Splicing is Common among Cyanobacterial Split Inteins: Implications for Protein Engineering

Department of Chemistry, Princeton University, 325 Frick Chemistry Laboratory, Princeton, New Jersey 08544, USA.
Journal of the American Chemical Society (Impact Factor: 12.11). 06/2012; 134(28):11338-41. DOI: 10.1021/ja303226x
Source: PubMed


We describe the first systematic study of a family of inteins, the split DnaE inteins from cyanobacteria. By measuring in vivo splicing efficiencies and in vitro kinetics, we demonstrate that several inteins can catalyze protein trans-splicing in tens of seconds rather than hours, as is commonly observed for this autoprocessing protein family. Furthermore, we show that when artificially fused, these inteins can be used for rapid generation of protein α-thioesters for expressed protein ligation. This comprehensive survey of split inteins provides indispensable information for the development and improvement of intein-based tools for chemical biology.

27 Reads
  • Source
    • "Protein splicing is a post-translational process catalyzed by a large family of proteins called inteins [56]. The split DnaE inteins family in cyanobacteria have been well studied, including the structure, distribution, splicing efficiencies and so on [40] [41] [42] [43] [44]. These comprehensive surveys of split inteins provide valuable information for the development of intein-based tools for chemical biology. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Cyanobacteria are a diverse group of Gram-negative bacteria and the only prokaryotes capable of oxygenic photosynthesis. Recently, cyanobacteria have attracted great interest due to their crucial roles in global carbon and nitrogen cycles and their ability to produce clean and renewable biofuels. To survive in various environmental conditions, cyanobacteria have developed a complex signal transduction network to sense environmental signals and implement adaptive changes. The post-translational modifications (PTMs) systems play important regulatory roles in the signaling networks of cyanobacteria. The systematic investigation of PTMs could contribute to the comprehensive description of protein species and to elucidate potential biological roles of each protein species in cyanobacteria. Although the proteomic studies of PTMs carried out in cyanobacteria were limited, these data have provided clues to elucidate their sophisticated sensing mechanisms that contribute to their evolutionary and ecological success. This review aims to summarize the current status of PTM studies and recent publications regarding PTM proteomics in cyanobacteria, and discuss the novel developments and applications for the analysis of PTMs in cyanobacteria. Challenges, opportunities and future perspectives in the proteomics studies of PTMs in cyanobacteria are also discussed.
    Journal of proteomics 08/2015; DOI:10.1016/j.jprot.2015.07.037 · 3.89 Impact Factor
  • Source
    • "The selected Ser+1 clones with b 10% spliced product had a flexible glycine residue at − 1 or − 2 in the N-extein and S(W/ Y)(P/C) in the C-extein. Both Tyr+2 and Trp+2 are preferred residues at this important position for splicing of the Npu DnaE intein when Cys+1 is present [11] [13] [19] [23] [24] [27] [32] [33]. The selection of amino acids known to affect protein structure (glycine in all Ser+1 selected clones and proline in 8 of 9 low-yield Ser+1 clones) suggests that slight changes in active-site geometry could contribute to the improved activity of the Npu DnaE intein with Ser+1. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Inteins self-catalytically cleave out of precursor proteins while ligating the surrounding extein fragments with a native peptide bond. Much attention has been lavished on these molecular marvels with the hope of understanding and harnessing their chemistry for novel biochemical transformations including coupling peptides from synthetic or biological origins and controlling protein function. Despite an abundance of powerful applications, the use of inteins is still hampered by limitations in our understanding of their specificity (defined as flanking sequences that permit splicing) and the challenge of inserting inteins into target proteins. We examined the frequently used Nostoc punctiforme Npu DnaE intein after the C-extein cysteine nucleophile (Cys+1) was mutated to serine or threonine. Previous studies demonstrated reduced rates and/or splicing yields with the Npu DnaE intein after mutation of Cys+1 to Ser+1. In this study, genetic selection identified extein sequences with Ser+1 that enabled the Npu DnaE intein to splice with only a 5-fold reduction in rate compared to the wild-type Cys+1 intein and without mutation of the intein itself to activate Ser+1 as a nucleophile. Three different proteins spliced efficiently after insertion of the intein flanked by the selected sequences. We then used this selected specificity to achieve traceless splicing in a targeted enzyme at a location predicted by primary sequence similarity to only the selected C-extein sequence. This study highlights the latent catalytic potential of the Npu DnaE intein to splice with an alternative nucleophile and enables broader intein utility by increasing insertion site choices. (C) 2014 MRC Laboratory of Molecular Biology. Published by Elsevier Ltd.
    Journal of Molecular Biology 11/2014; 426(24). DOI:10.1016/j.jmb.2014.10.025 · 4.33 Impact Factor
  • [Show abstract] [Hide abstract]
    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.
    Cellular and Molecular Life Sciences CMLS 08/2012; 70(7). DOI:10.1007/s00018-012-1120-4 · 5.81 Impact Factor
Show more