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Inteins are abundant in the microbial world and are often found within essential genes involved in DNA replication, recombination, and repair. Compelling examples of conditional protein splicing, where intein removal and subsequent host protein activation are highly dependent on environmental cues, suggest that some inteins serve as post\u2010translational regulatory elements. Within the cellular context, however, the factors influencing protein splicing are still largely unknown. In this work, we demonstrate that the splicing of an intein from\n                    <jats:italic>Mycobacterium smegmatis<\/jats:italic>\n                    DnaB, an essential helicase, is temperature sensitive. Using two artificial extein systems\u2014a\n                    <jats:italic>k<\/jats:italic>\n                    anamycin\n                    <jats:italic>i<\/jats:italic>\n                    ntein\n                    <jats:italic>s<\/jats:italic>\n                    plicing\n                    <jats:italic>r<\/jats:italic>\n                    eporter (KISR) and an in vitro splicing reporter\u2014we show that splicing is inhibited at elevated, yet physiological, temperatures. In accordance with our results from\n                    <jats:italic>M. smegmatis<\/jats:italic>\n                    , we find that overexpression of GroEL in\n                    <jats:italic>Escherichia coli<\/jats:italic>\n                    expressing the KISR system drastically increased antibiotic resistance in a temperature\u2010dependent manner. Under heat stress, we find that GroEL dramatically increases the levels of unspliced precursor protein. Mechanistically, GroEL does not appear to directly promote protein splicing, but rather increases the pool of unspliced precursor, which in turn results in more overall splicing. These findings represent the first description of a chaperone promoting protein splicing, demonstrating the ability of cellular factors to influence intein excision. From a physiological perspective, we hypothesize that a\n                    <jats:italic>M. smegmatis<\/jats:italic>\n                    DnaB intein may act as a switch to inhibit DNA replication under conditions of heat stress. Extending past this, our findings have broad implications for improving the efficiency of intein\u2010based protein engineering technologies.\n                  <\/jats:p>","DOI":"10.1002\/pro.70768","type":"journal-article","created":{"date-parts":[[2026,8,15]],"date-time":"2026-08-15T03:42:20Z","timestamp":1786765340000},"update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["A protein chaperone can stabilize the intein\u2010containing precursor to indirectly promote protein splicing"],"prefix":"10.1002","volume":"35","author":[{"given":"John S.","family":"Smetana","sequence":"first","affiliation":[{"name":"Department of Biological Sciences Murray State University  Murray Kentucky USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Christopher R.","family":"Powell","sequence":"additional","affiliation":[{"name":"Department of Biological Sciences Murray State University  Murray Kentucky USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Noy","family":"Bagdadi","sequence":"additional","affiliation":[{"name":"Department of Life Sciences Ben\u2010Gurion University of the Negev  Beer\u2010Sheva Israel"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Tia M.","family":"Ariagno","sequence":"additional","affiliation":[{"name":"Department of Biological Sciences Murray State University  Murray Kentucky USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Hazel","family":"Thomas","sequence":"additional","affiliation":[{"name":"Department of Biological Sciences Murray State University  Murray Kentucky USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"suffix":"II","given":"Joel","family":"Weinberger","sequence":"additional","affiliation":[{"name":"Department of Biological Sciences Murray State University  Murray Kentucky 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