A promiscuous split intein with expanded protein engineering applications

Abstract

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...

Chemical modification of proteins by insertion of synthetic peptides using tandem protein trans-splicing

“…In fact, many groups have repeatedly observed yields of 10% or less with ncAA incorporation into transporters 36 , ion channels [37][38][39] , and G protein-coupled receptors 40,41 . Although the generally low yields observed with tPTS likely restrict the approach to applications that do not require large amounts of protein, at least some of the above limitations can be addressed by engineering more promiscuous and efficient split inteins [10][11][12]42 or by adding affinity tags to promote split intein interactions 18 . Such improvements would allow the approach to be applied to a broader complement of target proteins.…”

Control ofϕC31 integrase-mediated site-specific recombination by protein trans splicing

“…Further characterization of their properties would make them available as orthogonal functional parts for regulation of serine integrase-based applications. More recently, mutations at residues within the GEP loop of the Npu DnaE split intein have been identified that affect the specificity, reaction rates, and tolerance to changes in the flanking residues 16,17 . Use of these intein variants with broader specificities would require minimum changes to the extein sequence of the target integrase, thereby reducing the potential risk of introducing deleterious mutations.…”

Dynamic structural order of a low complexity domain facilitates assembly of intermediate filaments

“…In order to study the conformation of the Tm1 tail domain in the context of fully assembled intermediate filaments, we used a split-intein system to produce a segmentally labeled form of the intact Tm1 protein (19). The tail domain, including residues 373 to 441, was expressed 40 separately from the N-terminus of the protein (residues 1 to 372).…”