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Inteins are proteins that excise themselves out of host proteins and ligate the flanking polypeptides in an auto-catalytic process called protein splicing. In nature, inteins are either contiguous or split. In the case of split inteins, the two fragments must first form a complex for the splicing to occur. Contiguous inteins have previously been artificially split in two fragments because split inteins allow for distinct applications than contiguous ones. Even naturally split inteins have been split at unnatural split sites to obtain fragments with reduced affinity for one another, which are useful to create conditional inteins or to study protein-protein interactions. So far, split sites in inteins have been heuristically identified. We developed Int&in, a web server freely available for academic research (https://intein.biologie.uni-freiburg.de) that runs a machine learning model using logistic regression to predict active and inactive split sites in inteins with high accuracy. The model was trained on a dataset of 126 split sites generated using the gp41-1, Npu DnaE and CL inteins and validated using 97 split sites extracted from the literature. Despite the limited data size, the model, which uses various protein structural features, as well as sequence conservation information, achieves an accuracy of 0.79 and 0.78 for the training and testing sets, respectively. We envision Int&in will facilitate the engineering of novel split inteins for applications in synthetic and cell biology., (© 2024 The Authors. Protein Science published by Wiley Periodicals LLC on behalf of The Protein Society.)

We recently developed the SiMPl plasmid toolbox, which is constituted by pairs of plasmids, generically indicated as pSiMPl x _N and pSiMPl x _C, which can be stably maintained in Escherichia coli with a single antibiotic x. The method exploits the split intein gp41-1 to reconstitute the enzyme conferring resistance toward the antibiotic x, whereby each enzyme fragment is expressed from one of the plasmids in the pair. pSiMPl plasmids are currently available for use with ampicillin, kanamycin, chloramphenicol, hygromycin, and puromycin. Here, we introduce another pair for use with spectinomycin/streptomycin, broadening the application spectrum of the SiMPl toolbox. To find functional splice sites in aminoglycoside adenylyltransferase, we apply a streamlined strategy looking exclusively at the flexibility of native cysteine and serine residues, which we first validated splitting the enzymes conferring resistance toward ampicillin, kanamycin, chloramphenicol, and hygromycin. This strategy could be used in the future to split other enzymes conferring resistance toward antibiotics., Competing Interests: The authors declare no competing financial interest., (© 2021 The Authors. Published by American Chemical Society.)