Protein Trans-Splicing of an Atypical Split Intein Showing Structural Flexibility and Cross-Reactivity
Bibliographic record
Abstract
Inteins catalyze a protein splicing reaction to excise the intein from a precursor protein and join the flanking sequences (exteins) with a peptide bond. In a split intein, the intein fragments (I(N) and I(C)) can reassemble non-covalently to catalyze a trans-splicing reaction that joins the exteins from separate polypeptides. An atypical split intein having a very small I(N) and a large I(C) is particularly useful for joining synthetic peptides with recombinant proteins, which can be a generally useful method of introducing site-specific chemical labeling or modifications into proteins. However, a large I(C) derived from an Ssp DnaX intein was found recently to undergo spontaneous C-cleavage, which raised questions regarding its structure-function and ability to trans-splice. Here, we show that this I(C) could undergo trans-splicing in the presence of I(N), and the trans-splicing activity completely suppressed the C-cleavage activity. We also found that this I(C) could trans-splice with small I(N) sequences derived from two other inteins, showing a cross-reactivity of this atypical split intein. Furthermore, we found that this I(C) could trans-splice even when the I(N) sequence was embedded in a nearly complete intein sequence, suggesting that the small I(N) could project out of the central pocket of the intein to become accessible to the I(C). Overall, these findings uncovered a new atypical split intein that can be valuable for peptide-protein trans-splicing, and they also revealed an interesting structural flexibility and cross-reactivity at the active site of this intein.
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How this classification was reachedexpand
Full frame machine prediction
Teacher imitationNot calibrated prevalence, not ground truth. Human validation pending. The Gemma side is a direct model label for every work in the frame, read from the title-only record. The Codex side is a classifier learned from the 10,348 direct Codex labels and calibrated to design-weighted sample rates; fields without enough sample support carry no Codex call. Candidate is the union of the two sides; consensus is their intersection. These outputs are machine_predicted_unvalidated and are not human labels.
Distilled classifier scores by category (both heads)
| Category | Codex | Gemma |
|---|---|---|
| Metaresearch | 0.000 | 0.000 |
| Meta-epidemiology (narrow) | 0.000 | 0.000 |
| Meta-epidemiology (broad) | 0.000 | 0.000 |
| Bibliometrics | 0.000 | 0.000 |
| Science and technology studies | 0.000 | 0.000 |
| Scholarly communication | 0.000 | 0.000 |
| Open science | 0.000 | 0.000 |
| Research integrity | 0.000 | 0.000 |
| Insufficient payload (model declined to judge) | 0.000 | 0.000 |
Machine scores (provisional)
The two teacher heads of the student model, read on this work. A score orders the frame for review; it never asserts a category, and the validation status ships verbatim with every row.
Baseline scores from an immature model (maturity gate not passed, 7 training rounds). Scores rank; they never assert a category.
score_only:v0-immature-baseline · verbatim from the scoring run: score_only means the number may rank works, and no category label ships from itClassification
machine, unvalidatedMachine predicted; a candidate call from one source (direct Gemma or distilled Codex), not a consensus.
How this classification was reached, model by model and score by score, is at the end of the page under "How this classification was reached".