Biological applications of protein splicing

Abstract

Protein splicing is a naturally occurring process in which a protein editor, called an intein, performs a molecular disappearing act by cutting itself out of a host protein in a traceless manner. In the two decades since its discovery, protein splicing has been harnessed for the development of several protein-engineering methods. Collectively, these technologies help bridge the fields of chemistry and biology, allowing hitherto impossible manipulations of protein covalent structure. These tools and their application are the subject of this Primer.

Figure 1. Mechanism of Protein Splicing

Protein splicing (A) and its variant protein trans-splicing (B).

Figure 2. Expressed Protein Ligation

The boxed region designated the native chemical ligation (NCL) reaction in which transthioesterification of the protein α-thioester by the N-terminal Cys polypeptide is followed by an S- to N-acyl shift to generate a new peptide bond linking the two polypeptides. α-thioesters can be obtained recombinantly, using engineered inteins, or by chemical synthesis. N-terminal Cys polypeptides can also be produced recombinantly or made using standard solid-phase peptide synthesis (SPPS) methods.

Figure 3. Examples of Proteins Modfiied by EPL and PTS

Expressed protein ligation (EPL) and protein trans-splicing (PTS) can be used site-specifically modify a wide variety of structurally and functionally diverse proteins, as the examples given in the figure illustrate. Modifications range from naturally-occurring post-translational modifications (PTMs) to unnatural moieties and include: A) D-amino acids (D-Ala), B) ester bonds, C) acetylated (N-acetyl-Lys) and D) methylated amino acids (N-tri-methyl-Lys), E) phospho-Ser/Thr, F) ubiquitination, G) isotopes (PET emitting ¹⁸F), H) fluorophores (fluorescein), I) photo-crosslinkers (photo-Met), J) Ser-ATP bi-substrate transition state analogues, K), β-turn mimics (nipecotic acid), L) photo-caging groups (photo-caged phospho-Ser), M) glycosylated and N) prenylated amino acids O) non-hydrolysable analogues of AMP and P) non-hydrolysable phosphomimics (Tyr phosphonate).

Figure 4. in vivo Applications of Protein Splicing

A) Schematic representation of intein mediated peptide or protein cyclization. The target polypeptide is expressed flanked by Int^C and Int^N at the N- and C-termini, respectively. Protein trans-splicing (PTS) results in the formation of a new peptide bond between the N- and C-termini of the target and thus generates a circularized peptide or protein. B) Control of protein splicing through ligand-induced intein complementation. The splicing activity of artificially split inteins can be controlled by fusion to exogenous auxiliary domains (in the figure, a ligand binding domain). A triggering event (in the figure, ligand binding) causes a conformational change in the auxiliary domain, which induces intein reconstitution and subsequent protein splicing.