Journal
FEBS JOURNAL
Volume 280, Issue 14, Pages 3256-3269Publisher
WILEY-BLACKWELL
DOI: 10.1111/febs.12307
Keywords
BIL domain; Hint domain; NMR spectroscopy; protein ligation; protein trans-splicing
Categories
Funding
- Viikki Doctoral Programme in Molecular Biosciences
- Academy of Finland [137995]
- Sigrid Juselius Foundation
- Biocenter Finland (NMR and mass-spectrometry facilities at the Institute of Biotechnology)
- National Doctoral Programme in Informational and Structural Biology
- Academy of Finland (AKA) [137995, 137995] Funding Source: Academy of Finland (AKA)
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Protein splicing in trans by split inteins has become a useful tool for protein engineering invivo and invitro. Inteins require Cys, Ser or Thr at the first residue of the C-terminal flanking sequence because a thiol or hydroxyl group in the side chains is a nucleophile indispensable for the trans-esterification step during protein splicing. Newly-identified distinct sequences with homology to the hedgehog/intein superfamily, called bacterial intein-like (BIL) domains, often do not have Cys, Ser, or Thr as the obligatory nucleophilic residue found in inteins. We demonstrated that BIL domains from Clostridiumthermocellum (Cth) are proficient at protein splicing without any sequence changes. We determined the first solution NMR structure of a BIL domain, CthBIL4, to guide engineering of split BIL domains for protein ligation. The newly-engineered split BIL domain could catalyze protein ligation by trans-splicing. Protein ligation without any nucleophilic residues of Cys, Ser and Thr could alleviate junction sequence requirements for protein trans-splicing imposed by split inteins and could broaden applications of protein ligation by protein trans-splicing.
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