Browsing by Subject "SPLIT INTEINS"

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  • Richardson, Dominique; Itkonen, Jaakko; Nievas, Julia; Urtti, Arto; Casteleijn, Marco G. (2018)
    The use of living cells for the synthesis of pharmaceutical proteins, though state-of-the-art, is hindered by its lengthy process comprising of many steps that may affect the protein’s stability and activity. We aimed to integrate protein expression, purification, and bioconjugation in small volumes coupled with cell free protein synthesis for the target protein, ciliary neurotrophic factor. Split-intein mediated capture by use of capture peptides onto a solid surface was efficient at 89–93%. Proof-of-principle of light triggered release was compared to affinity chromatography (His6 fusion tag coupled with Ni-NTA). The latter was more efficient, but more time consuming. Light triggered release was clearly demonstrated. Moreover, we transferred biotin from the capture peptide to the target protein without further purification steps. Finally, the target protein was released in a buffer-volume and composition of our choice, omitting the need for protein concentration or changing the buffer. Split-intein mediated capture, protein trans splicing followed by light triggered release, and bioconjugation for proteins synthesized in cell free systems might be performed in an integrated workflow resulting in the fast production of the target protein.
  • Beyer, Hannes M.; Mikula, Kornelia M.; Kudling, Tatiana V.; Iwaï, Hideo (2019)
    Self-splicing inteins are mobile genetic elements invading host genes via nested homing endonuclease (HEN) domains. All HEN domains residing within inteins are inserted at a highly conserved insertion site. A purifying selection mechanism directing the location of the HEN insertion site has not yet been identified. In this work, we solved the three-dimensional crystal structures of two inteins inserted in the cell division control protein 21 of the hyperthermophilic archaea Pyrococcus abyssi and Pyrococcus horikoshii. A comparison between the structures provides the structural basis for the thermo-stabilization mechanism of inteins that have lost the HEN domain during evolution. The presence of an entire extein domain in the intein structure from Pyrococcus horikoshii suggests the selection mechanism for the highly conserved HEN insertion point.
  • Beyer, Hannes M.; Iwai, Hideo (2019)
    Protein-splicing domains are frequently used engineering tools that find application in the in vivo and in vitro ligation of protein domains. Directed evolution is among the most promising technologies used to advance this technology. However, the available screening systems for protein-splicing activity are associated with bottlenecks such as the selection of pseudo-positive clones arising from off-pathway reaction products or fragment complementation. Herein, we report a stringent screening method for protein-splicing activity in cis and trans, that exclusively selects productively splicing domains. By fusing splicing domains to an intrinsically disordered region of the antidote from the Escherichia coli CcdA/CcdB type II toxin/antitoxin system, we linked protein splicing to cell survival. The screen allows selecting novel cis- and trans-splicing inteins catalyzing productive highly efficient protein splicing, for example, from directed-evolution approaches or the natural intein sequence space.
  • Oeemig, Jesper S.; Beyer, Hannes M.; Aranko, A. Sesilja; Mutanen, Justus; Iwai, Hideo (2020)
    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 -1 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 -1 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.