Highly Efficient and More General cis- and trans-Splicing Inteins through Sequential Directed Evolution
Bibliographic record
Abstract
Inteins are internal protein sequences that post-translationally self-excise and splice together the flanking sequences, the so-called exteins. Natural and engineered inteins have been used in many practical applications. However, inteins are often inefficient or inactive when placed in a non-native host protein and may require the presence of several amino acid residues of the native exteins, which will then remain as a potential scar in the spliced protein. Thus, more general inteins that overcome these limitations are highly desirable. Here we report sequential directed evolution as a new approach to produce inteins with such properties. Random mutants of the Ssp (Synechocystis sp. PCC 6803) DnaB mini-intein were inserted into the protein conferring kanamycin resistance at a site where the parent intein was inactive for splicing. The mutants selected for splicing activity were further improved by iterating the procedure for two more cycles at different positions in the same protein. The resulting improved inteins showed high activity in the positions of the first rounds of selection, in multiple new insertion sites, and in different proteins. One of these inteins, the M86 mutant, which accumulated 8 amino acid substitutions, was also biochemically characterized in an artificially split form with a chemically synthesized N-terminal intein fragment consisting of 11 amino acids. When compared with the unevolved split intein, it exhibited an ∼60-fold increased rate in the protein trans-splicing reaction and a Kd value for the interaction of the split intein fragments improved by an order of magnitude. Implications on the intein structure-function, practical application, and evolution are discussed. Inteins are internal protein sequences that post-translationally self-excise and splice together the flanking sequences, the so-called exteins. Natural and engineered inteins have been used in many practical applications. However, inteins are often inefficient or inactive when placed in a non-native host protein and may require the presence of several amino acid residues of the native exteins, which will then remain as a potential scar in the spliced protein. Thus, more general inteins that overcome these limitations are highly desirable. Here we report sequential directed evolution as a new approach to produce inteins with such properties. Random mutants of the Ssp (Synechocystis sp. PCC 6803) DnaB mini-intein were inserted into the protein conferring kanamycin resistance at a site where the parent intein was inactive for splicing. The mutants selected for splicing activity were further improved by iterating the procedure for two more cycles at different positions in the same protein. The resulting improved inteins showed high activity in the positions of the first rounds of selection, in multiple new insertion sites, and in different proteins. One of these inteins, the M86 mutant, which accumulated 8 amino acid substitutions, was also biochemically characterized in an artificially split form with a chemically synthesized N-terminal intein fragment consisting of 11 amino acids. When compared with the unevolved split intein, it exhibited an ∼60-fold increased rate in the protein trans-splicing reaction and a Kd value for the interaction of the split intein fragments improved by an order of magnitude. Implications on the intein structure-function, practical application, and evolution are discussed.
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How this classification was reachedexpand
Full frame distilled prediction
Teacher imitationNot calibrated prevalence, not ground truth. Human validation pending. Learned from the 10,348 direct Codex labels and 10,348 direct Gemma labels. Candidate is the union of thresholded teacher heads; consensus is their intersection. These outputs are machine_predicted_unvalidated and are not human labels or direct frontier model labels.
Codex and Gemma teacher scores by category
| 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 teacher head, 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".