Traceless protein splicing utilizing evolved split inteins

Abstract

Split inteins are parasitic genetic elements frequently found inserted into reading frames of essential proteins. Their association and excision restore host protein function through a protein self-splicing reaction. They have gained an increasingly important role in the chemical modification of proteins to create cyclical, segmentally labeled, and fluorescently tagged proteins. Ideally, inteins would seamlessly splice polypeptides together with no remnant sequences and at high efficiency. Here, we describe experiments that identify the branched intermediate, a transient step in the overall splicing reaction, as a key determinant of the splicing efficiency at different splice-site junctions. To alter intein specificity, we developed a cellbased selection scheme to evolve split inteins that splice with high efficiency at different splice junctions and at higher temperatures. Mutations within these evolved inteins occur at sites distant from the active site. We present a hypothesis that a network of conserved coevolving amino acids in inteins mediates these long-range effects. directed evolution ͉ protein ligation ͉ Crk-II ͉ SCA ͉ coevolution P rotein function is modulated by a variety of posttranslational modifications, such as phosphorylation, ubiquitylation, and methylation (1). One of the most dramatic posttranslational modifications is protein splicing, an autocatalytic process in which an intervening polypeptide sequence, termed an intein, is excised from a precursor protein with concomitant splicing of the franking sequences, known as exteins. Protein splicing has proven to be a highly versatile process for methods development and forms the hub of several protein engineering technologies (2). Inteins have been successfully used in protein semisynthesis to create posttranslationally modified proteins (2, 3), in the cyclization of peptides to create small molecule toxins (4), in plant biotechnology to reconstruct proteins in vivo (5), in the segmental labeling of proteins for NMR studies (6), and in protein semisynthesis in living cells (7).Inteins come in 2 flavors-cis splicing inteins are single polypeptides that are embedded in a host protein, whereas trans-splicing inteins (herein called split inteins) are separate polypeptides that mediate protein splicing after the intein pieces and their protein cargo associate (8, 9) (Fig. 1A). Despite the many applications of split inteins in chemical biology and protein chemistry, they are plagued with various idiosyncratic parameters that limit their more general use. Most importantly, the 2 parts of the naturally split inteins associate and typically splice at a C-terminal junction containing the canonical ''CFN'' tripeptide sequence (10, 11), which are the first 3 amino acids of the C-extein sequence (Fig. 1 A). This tripeptide remains in the product after splicing, meaning that it will most often be a mutant protein. It is currently unclear why this "CFN" sequence is required for efficient protein trans-splicing. In contrast, work from several laboratories, incl...

Structure of the branched intermediate in protein splicing

“…Rather, the linked exteins are perched on top of the intein, where they assume an unusual corkscrewlike structure that has minimal impact on the intein-fold. This arrangement is in keeping with the known promiscuity of inteins with respect to the flanking extein sequences, a property that is integral to their use in protein biotechnology (16,22). Of the conserved active-site residues in the intein, only two assume an altered conformation in the branched intermediate structure compared with the linear intein precursor, namely the block F His and the block G Asn.…”

Structural and Dynamical Features of Inteins and Implications on Protein Splicing

“…This single V67L mutation minimally affects the crystal structure (20) but has profound effects on global dynamics specifically on the dynamics of N and C termini of the intein shown by NMR chemical shift perturbations and H/D exchange (45). Directed evolution studies also show that distal mutations have enhancing effects on splicing (40,44,46). In the Ssp DnaB mini-intein, the mutations acquired through directed evolution have additive effects, and the mutant intein becomes more tolerant to the local extein sequences and the constraints these sequences impose on the intein active site (44).…”

“…In the Ssp DnaB mini-intein, the mutations acquired through directed evolution have additive effects, and the mutant intein becomes more tolerant to the local extein sequences and the constraints these sequences impose on the intein active site (44). Similarly, the Npu DnaE intein, selected against noncanonical C-extein (SGV instead of the native CFN), accumulate mutations distal from the active site (40). These mutations were identified as either a part of or juxtaposed to a spatially contiguous network of amino acids predicted to be energetically coupled to the active site.…”

“…For example, in the Npu DnaE intein, the splicing is minimally affected by local N-extein sequence, but the variation of the local C-extein sequence dramatically affects splicing efficiency (37-39) by perturbing branched intermediate resolution (40). In the structure of the orthologous Ssp DnaE intein, the native Pheϩ2 side chain packs against and stabilizes Block F histidine (Fig.…”

Mammalian synthetic biology: emerging medical applications

“…As inteins are transcribed and translated together with the target protein before they undergo autocatalytic self-excision and splicing, inteins increase the time it takes to transcribe, translate and posttranslationally process a target gene. The intein domain can be transcribed and translated by two separate genes (known as split inteins) and the resulting precursor proteins splice each other, process termed trans-splicing, to yield a single functional protein [106,107]. The protein splicing and trans-splicing processes can be triggered by small molecules or protein inputs to obtain intein-based post-translational switches [108][109][110][111].…”