Protein Semi-Synthesis in Living Cells

Giriat, Izabela; Muir, Tom W.

doi:10.1021/ja034736i

Cited by 162 publications

(115 citation statements)

References 14 publications

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“…The improved cyclization enabled by Cfa GEP may be further extended to a SICLOPPS library and selection system to identify cyclic peptides that bind or inhibit a target enzyme (23). Furthermore, the greater splicing yields demonstrated for histone semisynthesis with the Cfa GEP intein in nucleo could be applied both to histones and other cellular proteins in live cells (30,31). Beyond the applications demonstrated in this study, we would expect the engineered GEP loop mutation to also improve many other uses of naturally split inteins, including the generation of segmentally labeled proteins for NMR spectroscopy (32,33) and the production of recombinant proteins that would otherwise be incompatible with cellular expression systems (34).…”

Section: Discussionmentioning

confidence: 99%

A promiscuous split intein with expanded protein engineering applications

Stevens

Sekar

Shah

et al. 2017

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

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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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Section: Discussionmentioning

confidence: 99%

A promiscuous split intein with expanded protein engineering applications

Stevens

Sekar

Shah

et al. 2017

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

Self Cite

117

119

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“…Semi-synthetic protein trans-splicing emerges as a new methodology with potential applications for protein semi-synthesis (9,15,(27)(28)(29)(30) and for mechanistic studies of the proteinsplicing pathway. Specific advantages over other approaches like chemical ligation strategies are the inherent affinity and specific recognition of the intein fragments as well as the circumvention of the otherwise required chemical functional groups like a thioester, an azide, or a phosphine.…”

Section: Discussionmentioning

confidence: 99%

“…These features simplify the synthesis of the synthetic extein-intein fragments, which could be carried out in a regular peptide facility using standard Fmoc solid phase peptide chemistry. They also offer the potential for protein semi-synthesis in complex mixtures like a cell extract (28) or even a live cell (9,27). The price one has to pay for this is the requirement to append the intein fragments to the actual peptide or protein sequences to be joined, i.e.…”

Section: Discussionmentioning

confidence: 99%

“…These methods take advantage of the protein ester intermediates generated during protein splicing, which can be cleaved by thiolysis or hydrolysis, and the finding that inteins can in principle be inserted into a heterologous protein context, i.e. they are quite tolerant toward foreign extein sequences (5)(6)(7)9). Other more recently emerging exciting avenues for protein engineering approaches rely on protein trans-splicing.…”

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confidence: 99%

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Interaction Studies and Alanine Scanning Analysis of a Semi-synthetic Split Intein Reveal Thiazoline Ring Formation from an Intermediate of the Protein Splicing Reaction

Ludwig

Schwarzer²,

Mootz

2008

Journal of Biological Chemistry

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We recently reported an artificially split intein based on the Ssp DnaB mini-intein that consists of a synthetic N-terminal intein fragment (Int N ) and a recombinant C-terminal part (Int C ), which are 11 and 143 amino acids in length, respectively. This intein holds great promise for the preparation of semi-synthetic proteins by protein trans-splicing. In this work we synthesized a set of Int N peptide variants to investigate their structurefunction relationship with regard to fragment association and promotion of protein trans-splicing. A further truncation of the Int N sequence below 11 amino acids resulted in loss of activity, whereas C-terminal extensions were tolerated. Alanine scanning analysis identified three essential hydrophobic residues, whereas substitutions at other positions were tolerated. We developed assays to monitor association of Protein splicing is a process in which an internal protein segment, the intein, excises itself out of a precursor protein (1-4). The flanking sequences, referred to as N-and C-exteins (Ex N and Ex C ), 3 are concomitantly linked with a native peptide bond. This remarkable post-translational protein backbone rearrangement is catalyzed by the intein in a spontaneous, autocatalytic reaction without the need of any additional factors or energy sources. Protein splicing is initiated by the attack of the nucleophilic side chain of a Cys or Ser residue at the first position of the intein on its upstream peptide bond to generate a thioester or oxoester intermediate, respectively. In the second step of the pathway, the Ex N acyl group is transferred by a trans(thio)esterification on the side chain of a Cys, Ser, or Thr at the first position of the Ex C . The side chain amide group of an Asn residue at the ultimate position of the intein then attacks its own carbonyl group to form a succinimide and thereby to cleave the peptide bond between the intein and the Ex C . In the final reaction, the thio-or oxoester between the liberated Ex N -Ex C quickly rearranges to the thermodynamically more stable peptide bond. This canonical mechanism of protein splicing as well as deviations from it found in certain inteins have been extensively reviewed (1-4). Inteins are useful as self-cleavable tags for protein purification strategies (5). They also have found many applications in the fields of protein chemistry and protein semi-synthesis, as they provide a route to obtain recombinant protein thioesters and proteins with an N-terminal cysteine residue important for chemical ligation reactions like native chemical ligation (NCL) and expressed protein ligation (6 -8). These methods take advantage of the protein ester intermediates generated during protein splicing, which can be cleaved by thiolysis or hydrolysis, and the finding that inteins can in principle be inserted into a heterologous protein context, i.e. they are quite tolerant toward foreign extein sequences (5-7, 9). Other more recently emerging exciting avenues for protein engineering approaches rely on protein trans-splicing. Th...

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“…As we will show here, switch-elements can be composed of depsipeptide (also called O-peptide or O-acyl isopeptide) units and/or pseudoprolines (CPro). So far, acyl transfer reactions have been the subject of extensive mechanistic studies, 24 and their role in protein biosynthesis and splicing, 25,26 peptide synthesis and solubilization, [27][28][29][30] prodrug design [31][32][33][34][35][36] and native chemoselective ligation strategies [37][38][39] has found broad attention. Our focus over the last few years was directed toward the elaboration of the concept of ''switch-peptides'' for the study of peptide self-assembly, secondary structure formation, and disruption.…”

Section: The Concept Of Switch-peptidesmentioning

confidence: 99%