An Atypical Naturally Split Intein Engineered for Highly Efficient Protein Labeling

Abstract

Protein trans-splicing catalyzed by split inteins is a powerful technique for assembling a polypeptide backbone from two separate parts. However, split inteins with robust efficiencies and short fragments suitable for peptide synthesis are rare and have mostly been artificially created. The novel split intein AceL-TerL was identified from metagenomic data and characterized. It represents the first naturally occurring, atypically split intein. The N-terminal fragment of only 25 amino acids is the shortest natural intein fragment to date and was easily amenable to chemical synthesis with a fluorescent label. Optimal protein trans-splicing activity was observed at low temperatures. Further improved mutants were selected by directed protein evolution. The engineered intein variants with up to 50-fold increased rates showed unprecedented efficiency in chemically labeling of a diverse set of proteins. These inteins should prove valuable tools for protein semi-synthesis and other intein-related biotechnological applications.

Improved protein splicing using embedded split inteins

“…Moreover, in vivo activity screens of protein trans-splicing have significantly accelerated the discovery of constructs with improved splicing activities. 5,12,20,21 A comparable screen for intein expression yield, such as applying a GFP reporter, may prove to be similarly beneficial. 22 Given the improvements in expression yield and PTS efficiency reported in this study, and the potential for additional refinement, we expect the split intein embedding strategies will expand the utility of PTS-based technologies.…”

“…[3][4][5]7,11 In general, this type of information is not available for other naturally split inteins, which continue to be identified from metagenomic data. 6,12 More general strategies to enhance stability across both existing and yet to be discovered families of inteins would thus be beneficial. Herein, we offer two related approaches to this problem, both of which involve embedding the split intein within a protein sequence designed to stabilize either the intein fragment itself or the appended extein.…”

Cyclic Peptides Made by Linking Synthetic and Genetically Encoded Fragments

“…The liberated recombinant peptide part (SIEGSGG(AcF)H 6 , 4 ) was also observed, as a consequence of the C‐terminal cleavage activity of the intein (Figure C, D). This well‐known side reaction was significantly reduced (from 18 to 7 %) at a lower temperature (8 °C), in accordance with a previous report (Figure S2) . No linear form of the peptide (lack of bond formation by either protein trans ‐splicing or oxime ligation) was observed, thus indicating quantitative conversion of the spliced material to the cyclic product (yields in Table S3).…”

Off‐Pathway‐Sensitive Protein‐Splicing Screening Based on a Toxin/Antitoxin System

“…The unique autocatalytic reaction catalyzed by inteins, which is capable of ligating protein domains, has enabled diverse biotechnological applications of inteins such as protein purification, ligation, and chemical modification . Protein splicing can not only occur in cis but also in trans , through artificially or naturally split inteins, leading to the ligation of individual polypeptides.…”