The first notable application of PTS was in 1998, when Yamazaki et al demonstrated a segmental isotopic labeling for NMR spectroscopy using split inteins (Yamazaki et al., 1998). Since then, the variety of applications has expanded vastly: PTS has been utilized in production of cytotoxic proteins (Wu et al., 2002), transgenic plants (Yang et al., 2003), protein cyclization (Charles P Scott et al., 1999) , protein switches (Buskirk and Liu, 2005) and  in vivo and in vitro protein engineering (Aranko et al., 2013).  

For site-specific protein modification and labeling, a very useful method is to splice proteins with synthetic peptides employing atypical split inteins. This is because the synthetic peptide can be designed to contain a diverse choice of unnatural amino acids and chemical modifications. (Lin et al., 2013)  

Protein purification vectors

Inteins can be used in affinity purification, first developed by (Chong, Shaorong et al., 1997)When used for protein purification, the intein’s cleavage activity must be suppressed in vivo, but still remain inducible in vitro. By mutating the the C-terminal Asn and the + 1aa, the cleavage reaction is limited to step 1 of the standard splicing pathway, and the cleavage at the N-terminal splice junction is easily controlled. (Chong, S. et al., 1998)  

Furthermore, for a intein-mediated affinity purification to be successful, an affinity tag must be added, as first presented by (LaVallie and McCoy, 1995). 

The 1st step of the splicing pathway ends in the forming of a thioester bond, which is usually stable until undergoing a nucleophilic attack, for example a by thiol reagent (David et al., 2004). While other thiol reagents are quick to hydrolyze from the freed target protein’s C-terminus, other have a more stable linkage.  The C-terminal thioester can be used to ligate various types of molecules to the target protein’s C-terminus. (Chong, Shaorong et al., 1998)  

TWIN or two-intein system is a technique used to produce cyclized peptides, and even proteins as large as 395 amino acids. The TWIN system places a C-terminus cleaving intein in the other end of the target protein, and an intein modified to undergo a thiol-induced N-terminal cleavage. When the target protein is cleaved at the C-terminus by the former intein, the result is an N-terminal Cys. When cleaved by the latter intein thiol-inducedly, a C-terminal thioester is produced on the same target protein. (Evans et al., 2001)  After the cleavage reactions, these two reactive groups react in a spontaneous condensation (Tam et al., 1995).  

SICLOPPS is an abbreviation of split intein-mediated circular ligation of peptides and proteins, which is a method to splice proteins in trans and cyclize the target protein (Charles P Scott et al., 1999).   

In vivo protein engineering

Salt-inducible protein engineering

An example of engineering: a simplified schematic drawing ofprotein cyclization utilizing salt-inducible inteins 

Protein trans-splicing (PTS) allows segmental isotopic labeling in much less time than expressed protein ligation (EPL) method and has a better yield (Minato et al., 2012). Moreover, since individual expression and purification steps are not required when engineering proteins in vivo, it consumes less time when compared to in vitro (Züger and Iwai, 2005), as the in vitro approach requires the bacterial expression host to be grown in an isotope-enriched culture medium (Xu, R. et al., 1999).  

Integrating an unlabeled tag into to a isotopically labeled target protein, the solubility and stability of the protein increases, and can be analyzed in NMR spectroscopy. This enables incorporating enhancement tags to otherwise unstable or soluble constructs. (Züger and Iwai, 2005

Protein splicing in trans allows the construction of site-specific reconstruction tools which can be utilized in at protein level in vivo. Very suitable for this use is a group of site-specific recombinases called the serine integrases. (Stark, 2017)  They were originally found from bacteriophages, and are short, very directional in recombination, and simple in their site requirements (Stark, 2014).  

A split-intein фC31-serine integrase was designed and constructed by using intein-mediated splicing to fuse together two precursor elements (Olorunniji et al., 2019).  During the process, the фC31 integrase was split at the non-conserved region of the recombinase domain, and then two well-characterised split-intein components were edited and attached to the integrase sequences. According to the research done by Olorunniji et al in 2019, splicing in trans is essential for the recombinase activity. 

This new split-intein regulated фC31-serine integrase can be utilized as a tool for transient inversion of genetic regulatory modules and could be the means to achieve a fast and accurate switching in enzyme activity in vivo. (Olorunniji et al., 2019

In addition, if developed further, this technique could be applied on building enhanced computational and memory systems in cell, such as orthogonal logic gate components (Maranhao and Ellington, 2013). They in turn could be utilized in conditional gene expression regulation, opening up many possibilities for genome engineering applications (Roquet et al., 2016).  

Gene therapy

Gene therapy is the medical field and practice of using therapeutic delivery of nucleic acid into patient’s cells as a treatment to a disease (Mulligan, 1993).  Typical vectors for gene transfer are adenovirus and adeno-associated virus (AAV), however, even when with these frequently used vectors, the procedure is not completely unhindered. Inteins and protein splicing they mediate could be beneficial in achieving  precise targeted and controlled delivery of important proteins. (Cheriyan and Perler, 2009)  

Basics behind PTS-based gene therapy

An example of intein-based gene therapy: expression of large genes utilizing PTS

With adenovirus-based vectors, especially when treating patients with cancer, reducing the toxicity is essential, and this can be achieved with identifying how the target gene is delivered to its specific site. For adenovirus to integrate into human cells, it’s capsid fiber protein must be recognized by a coxsackie-Ad receptor (CAR) on the human cell’s surface. (Bergelson et al., 1997)  The CARs can be found on most human cell types, but to enhance and develop viral targeting systems, CAR D1 domain has been modified using expressed protein ligation (EPL). A modified adapter molecule, incubated with adenovirus, was added to the cells, and the adapters were reported to redirect viral transduction with high efficiency to cells expressing matching receptors. (Nyanguile et al., 2003)    

In designing vectors, AAV is a very suitable option, since it is non-pathogenic, rarely induces any immune responses in patients, and its genomic incorporation site is well known and characterized (Kremer and Perricaudet, 1995).  

However, the DNA packaging size is very limited, and split inteins have been presented as one of the solutions for this obstacle. In an experiment was demonstrated that muscular dystrophy symptoms can be treated in the mdx mouse model by infecting with two AAV vectors that are each carrying halves of a large Becker-form dystrophin gene, with intein-coding sequences incorporated within.  This result suggests that other large proteins could be transfected into target cells with the same technique. (Li et al., 2008)  

Using splicing in trans is more favorable when compared to other types of split gene delivery involving gene reconstruction; trans-splicing is orientation specific in nature. Furthermore, this method can be transferred to other viral vectors with larger DNA capacity and therefore widening the range of diseases treatable by gene therapy. (Cheriyan and Perler, 2009)