Unprecedented Rates and Efficiencies Revealed for New Natural Split Inteins from Metagenomic Sources

Carvajal-Vallejos, Patricia K.; Pallisse, Roser; Mootz, Henning D.; Schmidt, Stefan

doi:10.1074/jbc.m112.372680

Cited by 142 publications

(190 citation statements)

References 51 publications

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“…This reaction was abrogated in a control construct with a C1A mutation, which exhibited only slow C-cleavage (Fig. 9b, left panel), consistent with an earlier study (27). On the other hand, a Sϩ1C mutation to generate the Cys1/Cysϩ1 combination resulted only in a little decrease of splicing efficiency.…”

Section: Journal Of Biological Chemistry 28799supporting

confidence: 73%

“…One reason to test this intein was the CSXXH signature in its block B/N3 motif analogous to the GOS-TerL intein. Furthermore, the gp41-1 intein is the split intein with the highest kinetic rate reported so far (27). Thus, residual activity with a Ser1/Cysϩ1 combination should be observable more easily than with other inteins.…”

Section: Journal Of Biological Chemistry 28799mentioning

confidence: 99%

“…The AceL-TerL intein consists of a short Int N fragment of 25 aa and a C-terminal fragment of 104 aa (25). Metagenomic databases proved already earlier to be a rich source for new split inteins with interesting properties (26,27). For example, the gp41-1 intein is the fastest splicing split intein described so far (27).…”

mentioning

confidence: 99%

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An Unprecedented Combination of Serine and Cysteine Nucleophiles in a Split Intein with an Atypical Split Site

Bachmann

Mootz

2015

Journal of Biological Chemistry

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Background: Intein-mediated protein splicing involves rearrangement of the polypeptide backbone through ester intermediates and excises the intein from the precursor. Results: An unusual split intein was identified that converts an oxoester into a thioester. Conclusion: Inteins can catalyze two consecutive formally uphill reactions. Significance: The first example of this special case in protein splicing underlines the mechanistic diversity of inteins.

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Section: Journal Of Biological Chemistry 28799supporting

confidence: 73%

Section: Journal Of Biological Chemistry 28799mentioning

confidence: 99%

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An Unprecedented Combination of Serine and Cysteine Nucleophiles in a Split Intein with an Atypical Split Site

Bachmann

Mootz

2015

Journal of Biological Chemistry

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“…S1B). Unlike many well-characterized contiguous inteins that splice slowly, several naturally split inteins demonstrate rapid splicing kinetics (3)(4)(5)(6). Indeed, the discovery of these ultrafast split inteins has enabled the development of numerous tools for both synthetic and biological applications (1).…”

mentioning

confidence: 99%

“…Accordingly, there is a need for split inteins whose activities are minimally affected by local sequence environment. Although efforts have previously been made to engineer promiscuous inteins (12,13), these have not focused on naturally split inteins, which have superior fragment association and splicing kinetics (4)(5)(6).…”

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confidence: 99%

A promiscuous split intein with expanded protein engineering applications

Stevens

Sekar

Shah

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

109

119

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The protein trans-splicing (PTS) activity of naturally split inteins has found widespread use in chemical biology and biotechnology. However, currently used naturally split inteins suffer from an "extein dependence," whereby residues surrounding the splice junction strongly affect splicing efficiency, limiting the general applicability of many PTS-based methods. To address this, we describe a mechanism-guided protein engineering approach that imbues ultrafast DnaE split inteins with minimal extein dependence. The resulting "promiscuous" inteins are shown to be superior reagents for protein cyclization and protein semisynthesis, with the latter illustrated through the modification of native cellular chromatin. The promiscuous inteins reported here thus improve the applicability of existing PTS methods and should enable future efforts to engineer promiscuity into other naturally split inteins. A n intein is an intervening protein domain that undergoes a unique posttranslational autoprocessing event, termed protein splicing. In this spontaneous process, the intein excises itself from the host protein and, in the process, ligates together the flanking N-and C-terminal residues (exteins) to form a native peptide bond (SI Appendix, Fig. S1A) (1, 2). Although inteins are most frequently found as a contiguous domain, some exist in a naturally split form. In this case, the two fragments are expressed as separate polypeptides and must associate before splicing takes place, so-called protein trans-splicing (SI Appendix, Fig. S1B). Unlike many well-characterized contiguous inteins that splice slowly, several naturally split inteins demonstrate rapid splicing kinetics (3-6). Indeed, the discovery of these ultrafast split inteins has enabled the development of numerous tools for both synthetic and biological applications (1).A major caveat to splicing-based methods is that all characterized inteins exhibit a sequence preference at extein residues adjacent to the splice site. In addition to a mandatory catalytic Cys, Ser, or Thr residue at position +1 (i.e., the first residue within the C-extein), there is a bias for residues resembling the proximal N-and C-extein sequence found in the native insertion site. Deviation from this preferred sequence context leads to a marked reduction in splicing activity, limiting the applicability of protein trans-splicing (PTS)-based methods (3, 7-11). Accordingly, there is a need for split inteins whose activities are minimally affected by local sequence environment. Although efforts have previously been made to engineer promiscuous inteins (12, 13), these have not focused on naturally split inteins, which have superior fragment association and splicing kinetics (4-6).Here, we report engineered versions of naturally split inteins that possess greatly improved extein tolerance. Guided by our understanding of active site interactions critical for efficient protein splicing, we carried out targeted saturation mutagenesis of an ultrafast split intein and then used a cell-based selection system to...

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Substrate specificities of inteins investigated by QuickDrop‐cassette mutagenesis

et al. 2020

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Inteins catalyze self-excision from host precursor proteins while concomitantly ligating the flanking substrates (exteins) with a peptide bond. Noncatalytic extein residues near the splice junctions, such as the residues at the À1 and +2 positions, often strongly influence the protein-splicing efficiency. The substrate specificities of inteins have not been studied for many inteins. We developed a convenient mutagenesis platform termed "QuickDrop"-cassette mutagenesis for investigating the influences of 20 amino acid types at the À1 and +2 positions of different inteins. We elucidated 17 different profiles of the 20 amino acid dependencies across different inteins. The substrate specificities will accelerate our understanding of the structure-function relationship at the splicing junctions for broader applications of inteins in biotechnology and molecular biosciences.

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Unprecedented Rates and Efficiencies Revealed for New Natural Split Inteins from Metagenomic Sources

Cited by 142 publications

References 51 publications

An Unprecedented Combination of Serine and Cysteine Nucleophiles in a Split Intein with an Atypical Split Site

An Unprecedented Combination of Serine and Cysteine Nucleophiles in a Split Intein with an Atypical Split Site

A promiscuous split intein with expanded protein engineering applications

Substrate specificities of inteins investigated by QuickDrop‐cassette mutagenesis

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