Protein trans‐splicing on an M13 bacteriophage: towards directed evolution of a semisynthetic split intein by phage display

Abstract

Split inteins link their fused peptide or protein sequences with a peptide bond in an autocatalytic reaction called protein trans-splicing. This reaction is becoming increasingly important for a variety of applications in protein semisynthesis, polypeptide circularisation, construction of biosensors, or segmental isotopic labelling of proteins. However, split inteins exhibit greatly varying solubility, efficiency and tolerance towards the nature of the fused sequences as well as reaction conditions. We envisioned that phage display as an in vitro selection technique would provide a powerful tool for the directed evolution of split inteins with improved properties. As a first step towards this goal, we show that presentation of active split inteins on an M13 bacteriophage is feasible. Two different C-terminal intein fragments of the Ssp DnaB intein, artificially split at amino acid positions 104 and 11, were encoded in a phagemid vector in fusion to a truncated gpIII protein. For efficient production of hybrid phages, the presence of a soluble domain tag at their N-termini was necessary. Immunoblot analysis revealed that the hybrid phages supported protein trans-splicing with a protein or a synthetic peptide, respectively, containing the complementary intein fragment. Incorporation of biotin or desthiobiotin by this reaction provides a straightforward strategy for future enrichment of desired mutants from randomised libraries of the C-terminal intein fragments on streptavidin beads. Protein semisynthesis on a phage could also be exploited for the selection of chemically modified proteins with unique properties.

Bacterial Cell‐Surface Display of Semisynthetic Cyclic Peptides

“…We first tested the integrity and activity of Int C displayed without the unnatural amino acid AcF (Figure ). A single nonconserved cysteine in the Int C fragment was removed by site‐directed mutagenesis (C50S) to avoid potential complications caused by unspecific disulfide formation . Cells presenting the Int C ‐AIDA‐I fusion protein ( 1 ) were washed and resuspended in splicing buffer (pH 7; see Figure S1 in the Supporting Information for expression of 1 ; see Table S2 for all sequences).…”

Evolving Accelerated Amidation by SpyTag/SpyCatcher to Analyze Membrane Dynamics

“…Selection from phage libraries has been established for decades and the difficult thing is usually to detect weak interactions,4 rather than the challenge of screening for irreversible interactions 1b, 5. We established a panning procedure to select for covalent bond formation between SpyTag variants and the SpyCatcher bait (Figure 1 a, see Supporting Information for detailed methods).…”

“…However,e ngineering covalent interactions between protein partners brings ar ange of new opportunities for basic research and synthetic biology. [1] We have developed the use of spontaneous amide bond formation by peptide tags as asimple,specific, and genetically-encoded route to lock protein units together. [2] This technology, particularly the SpyTag/SpyCatcher pair, has been used in diverse applications including biomaterials,n ext-generation sequencing,e nzyme stabilization, and vaccine development.…”

Highly Efficient and More General cis- and trans-Splicing Inteins through Sequential Directed Evolution

“…After protein splicing, these native extein residues would remain in the spliced protein, which may affect the function of the spliced protein and thus constrain where the intein can be inserted. Directed protein evolution has been used previously for engineering inteins, either by using an in vivo selection based on the reconstitution of a selectable protein through splicing (7, 28 -32) or by exploiting in vitro phage display systems (33,34). However, in these examples, either the flanking amino acids at the splice junctions have been kept constant or only a single site was used.…”