Author-  Abhishek Kumar ,Integrated MSc, SBS, NISER, Bhubaneswar, India , Content Editor (Biotechticle.com)

Protein splicing is a post-translational modification in which a stretch of amino acids (intein) flanked by two exteins is excised. Unlike RNA splicing which requires additional enzymes intein excises itself and joins the exteins. The precursor prior to splicing contains an N-extein followed by the central intein followed by a C-extein [1]. Inteins have been described as in-frame intervening sequences that produce exteins [2]. A online database of all inteins has been prepared and is called as Inbase.

The N-terminal residue of intein can be serine, cysteine or threonine these attack the preceding peptide bond to form an ester or thioester. The C-terminal must be an asparagine, which undergoes cyclization to cleave the peptide bond and release intein and extein. At last in the extein the ester or thioester bond is rearranged to form a normal peptide bond [3].

Inteins can be broadly divided into 4 types :

1. Maxi-intein: N and C terminal splicing domain with an endonuclease domain
2. Mini-intein: N and C terminal splicing domain without an endonuclease domain.
3. Trans-splicing intein: Split into 2 or more domains[4]
4. Alanine intein: Has alanine instead of serine and cysteine

The endonuclease gene codes for a homing endonuclease enzyme that can transpose the intein coding DNA to a previously intein-free site. The recognition site for homing endonuclease are very long. So, the probability of finding these sites randomly is very low [5, 6]. Inteins have also been called as selfish genes (For more details you can see the biotechticle post on selfish genes). Since, they generally do not make any contribution to the reproductive success of the host [6]. Inteins are found in all three domains of life: Archaea, bacteria and Eukaryotes. The distribution of inteins and exteins vary greatly among different species and hosts. These are still not known to be present in metazoans and plants. Inteins are difficult to detect, so, probability of presence of inteins in animal genome still exists [7].

Applications in Biotechnology:

• Protein semi-synthesis/ Expressed protein ligation (EPL): Inteins are used to incorporate in-vitro unnatural amino acids, probes and post-translational modifications after expression. This has given new insights about protein folding, enzyme mechanisms, ion-channel function etc. [8].
• Mitochondrial gene therapy: It is very difficult for import of mitochondrial proteins from cytosol due to their high hydrophobicity. So, transgenes are added to reduce the hydrophobicity of proteins which can be later spliced out from the proteins by a method called as post-trans splicing (PTS) [9].
• Isotope labelling: Proteins can be fitted with different isotope labelling schemes to generate simple and distinct NMR spectra. This can be assembled in vitro or in vivo [10].
• Self-aggregating peptides: Some proteins called elastin-like polypeptides can cause proteins to form aggregates. When fused with a target protein they can form aggregates inside the cells which can used for protein isolation in a continuous media flow yielding high amounts of proteins [11].
• Removal of affinity tags: Affinity tags are used to purify the protein of interest from the cell. Sometimes it is necessary to remove the affinity tags after purification. Using proteases can cause non-specific proteolysis. This can be avoided by removing the affinity tag after purification using intein based protein splicing.

References:

1. Anraku, Y; Mizutani, R; Satow, Y (2005). “Protein splicing: its discovery and structural insight into novel chemical mechanisms”. IUBMB Life. 57 (8): 563–74.
2. Perler FB; Davis EO; Dean GE; Gimble FS; Jack WE; Neff N; Noren CJ; Thorner J; Belfort M (1994) Protein splicing elements: inteins and exteins–a definition of terms and recommended nomenclature. Nucleic Acids Res. Apr 11; 22(7): 1125-7.
3. Perler FB; Noren CJ; Wang J (2000) Dissecting the Chemistry of Protein Splicing and Its Applications. Angew. Chem. Int. Ed. 39: 450 – 466.
4. Wu, H.; Hu, Z.; Liu, X. Q. (1998). “Protein trans-splicing by a split intein encoded in a split DNA-E gene of Synechocystis sp. PCC6803”. Proceedings of the National Academy of Sciences of the United States of America. 95 (16): 9226–9231.
5. Jasin M (Jun 1996). “Genetic manipulation of genomes with rare-cutting endonucleases”. Trends Genet. 12 (6): 224–8. doi:10.1016/0168-9525(96)10019-6.
6. Doolittle WF; Sapienza C (1980). “Selfish genes, the phenotype paradigm and genome evolution”. Nature. 284 (5757): 601–603.
7. Pietrokovski S (1998). “Identification of a virus intein and a possible variation in the protein-splicing reaction.” Current Biology 8(18): 634-638.
8. Schwarzer D; Cole PA (2005). “Protein semi-synthesis and expressed protein ligation: chasing a protein’s tail”. Curr Opin Chem Biol. 9 (6): 561–9.
9. De Grey; Aubrey D.N.J (2000). “Mitochondrial gene therapy: an arena for the biomedical use of inteins”. Trends in Biotechnology. 18 (9): 394–399.
10. Züger S; Iwai H (2005) “Intein-based biosynthetic incorporation of unlabelled protein tags into isotopically labeled proteins for NMR studies.” Nat Biotechnol. 2005 Jun; 23(6):736-40.
11. Lei X.; Wei W.; Bihong W.; Zhanglin L. (2011). “Streamlined protein expression and purification using cleavable self-aggregating tags”. Microbial Cell Factories. 10 (1): 42.
12. Chong, S. et al (1997). “Single-column purification of free recombinant proteins using a self-cleavable affinity tag derived from a protein splicing element”. Gene. 192 (2)