Intein

The mechanism of protein splicing involving inteins. In this scheme, the N-extein is shown in red, the intein in black, and the C-extein in blue. X represents either an oxygen or sulfur atom.

An intein is a segment of a protein that is able to excise itself and join the remaining portions (the exteins) with a peptide bond in a process termed protein splicing. Inteins have also been called "protein introns".^[1]

Intein-mediated protein splicing occurs after the intein-containing mRNA has been translated into a protein. This precursor protein contains three segments — an N-extein followed by the intein followed by a C-extein. After splicing has taken place, the resulting protein contains the N-extein linked to the C-extein; this splicing product is also termed an extein.

History

The first intein was discovered in 1988 through sequence comparison between the Neurospora crassa^[2] and carrot^[3] vacuolar ATPase (without intein) and the homologous gene in yeast (with intein) that was first described as a putative calcium ion transporter.^[4] In 1990 Hirata et al.^[5] demonstrated that the extra sequence in the yeast gene was transcribed into mRNA and removed itself from the host protein only after translation. Since then, inteins have been found in all three domains of life (eukaryotes, bacteria, and archaea) and in viruses.

Most reported inteins also contain an endonuclease domain that plays a role in intein propagation. In fact, many genes have unrelated intein-coding segments inserted at different positions. For these and other reasons, inteins (or more properly, the gene segments coding for inteins) are sometimes called selfish genetic elements, but it may be more accurate to call them parasitic. According to Dawkins' gene centered view of evolution,^[6] all genes are selfish, even those that cooperate with other genes to build the organisms that carry the genes into the next generation. Most genes are "selfish" only insofar as to compete with other genes or alleles but usually they fulfill a function for the organisms, whereas "parasitic genetic elements", at least initially, do not make a positive contribution to the fitness of the organism.^[7]

Within the database of all known inteins (Inbase), 113 known inteins are present in eukaryotes with minimum length of 138 amino acids and maximum length of 844 amino acids. The first intein was found encoded within the VMA gene of Saccharomyces cerevisiae. They were later found in fungi (ascomycetes, basidiomycetes, zygomycetes and chytrids) and in diverse proteins as well. A protein distantly related to known inteins containing protein, but closely related to metazoan hedgehog proteins, has been described to have the intein sequence from Glomeromycota. Many of the newly described inteins contain homing endonucleases and some of these are apparently active (F. B. Perler, 2002). The abundance of intein in fungi indicates lateral transfer of intein-containing genes. While in eubacteria and archea, there are 289 and 182 known inteins until this literature review was written. Not surprisingly, most intein in eubacteria and archea are found to be inserted into nucleic acid metabolic protein, like fungi (F. B. Perler, 2002).

Mechanism

The mechanism for the splicing effect is a naturally occurring analogy to the technique for chemically generating medium-sized proteins called native chemical ligation, which was developed at the same time as inteins were discovered.^{[citation needed]}

The process begins with an N-O or N-S shift when the side chain of the first residue (a serine, threonine, or cysteine) of the intein portion of the precursor protein nucleophilically attacks the peptide bond of the residue immediately upstream (that is, the final residue of the N-extein) to form a linear ester (or thioester) intermediate. A transesterification occurs when the side chain of the first residue of the C-extein attacks the newly formed (thio)ester to free the N-terminal end of the intein. This forms a branched intermediate in which the N-extein and C-extein are attached, albeit not through a peptide bond. The last residue of the intein is always an asparagine, and the amide nitrogen atom of this side chain cleaves apart the peptide bond between the intein and the C-extein, resulting in a free intein segment with a terminal cyclic imide. Finally, the free amino group of the C-extein now attacks the (thio)ester linking the N- and C-exteins together. An O-N or S-N shift produces a peptide bond and the functional, ligated protein.^[8]

Inteins in biotechnology

Inteins are very efficient at protein splicing, and they have accordingly found an important role in biotechnology. There are more than 200 inteins identified to date; sizes range from 100–800 AAs. Inteins have been engineered for particular applications such as protein semisynthesis^[9] and the selective labeling of protein segments, which is useful for NMR studies of large proteins.^[10]

Pharmaceutical inhibition of intein excision may be a useful tool for drug development; the protein that contains the intein will not carry out its normal function if the intein does not excise, since its structure will be disrupted.

It has been suggested that inteins could prove useful for achieving allotopic expression of certain highly hydrophobic proteins normally encoded by the mitochondrial genome, for example in gene therapy (de Grey 2000). The hydrophobicity of these proteins is an obstacle to their import into mitochondria. Therefore, the insertion of a non-hydrophobic intein may allow this import to proceed. Excision of the intein after import would then restore the protein to wild-type.

The use of an affinity tag to purify recombinant proteins has been widely used as it allows accumulation of recombinant protein with little impurities. Though this is the case but affinity tag must be removed by protease in final step. The extra step of proteolysis raises problem of specificity of protease to remove affinity tag from recombinant protein. Also its digestion product has to be removed. This problem can be avoided by fusing affinity tag to self cleavable intein in controlled environment. The first generation of expression vector of this kind used modified Saccharomyces cerevisiae VMA (Sce VMA) intein. Chong et al. (1997) used chitin binding domain form Bacillus circulans as affinity tag and fused this tag in modified Sce VMA intein. The modified intein undergoes self-cleavage reaction at its N-terminal peptide linkage by 1,4-dithiothreitol (DTT), β-mercaptoethanol (β-ME) or cystine at low temperatures over a broad pH range. After expression of the recombinant protein cell homogenate is passed through the column containing chitin. This allows CBD of the chimeric protein to bind to column. Further when temperature is lowered and the above described molecules are passes through column, the chimeric protein undergoes self splicing and only target protein is eluted. This novel technique eliminated proteolysis step and modified Sce VMA stays in column attached to chitin through CBD (Chong et al., 1997).

Recently inteins have been used to purify proteins based on self aggregating peptides. Elastin-like polypeptides (ELPs) are a useful tool in biotechnology. Fused with target protein, they tend to form aggregates inside the cells (Fong & Wood, 2010). This eliminates the chromatographic step needed in protein purification. The ELP tags have been used in the fusion protein of intein, so that the aggregates can be isolated without chromatography (by centrifugation) and then intein and tag can be cleaved in controlled manner to release the target protein into solution. This protein isolation can be done using continuous media flow, yielding high amounts of protein, making this process more economically efficient than conventional methods (Fong & Wood, 2010). Another group of researchers used smaller self aggregating tags to isolate target protein. Small amphipathic peptides 18A and ELK16 (figure 5) were used to form self cleaving aggregating protein (Xing et al., 2011).

Intein naming conventions

The first part of an intein name is based on the scientific name of the organism in which it is found, and the second part is based on the name of the corresponding gene or extein. For example, the intein found in Thermoplasma acidophilum and associated with Vacuolar ATPase subunit A (VMA) is called "Tac VMA".

Normally, as in this example, just three letters suffice to specify the organism, but there are variations. For example, additional letters may be added to indicate a strain. If more than one intein is encoded in the corresponding gene, the inteins are given a numerical suffix starting from 5′ to 3′ or in order of their identification (for example, "Msm dnaB-1").

The segment of the gene that encodes the intein is usually given the same name as the intein, but to avoid confusion the name of the intein proper is usually capitalized (e.g., Pfu RIR1-1), whereas the name of the corresponding gene segment is italicized (e.g., Pfu rir1-1).

Full and mini inteins

Inteins can contain a homing endonuclease gene (HEG) domain in addition to the splicing domains. This domain is responsible for the spread of the intein by cleaving DNA at an intein-free allele on the homologous chromosome, triggering the DNA double-stranded break repair (DSBR) system, which then repairs the break, thus copying the intein-coding DNA into a previously intein-free site. The HEG domain is not necessary for intein splicing, and so it can be lost, forming a minimal, or mini, intein. Several studies have demonstrated the modular nature of inteins by adding or removing HEG domains and determining the activity of the new construct.^{[citation needed]}

Split inteins

Sometimes, the intein of the precursor protein comes from two genes. In this case, the intein is said to be a split intein. For example, in cyanobacteria, DnaE, the catalytic subunit α of DNA polymerase III, is encoded by two separate genes, dnaE-n and dnaE-c. The dnaE-n product consists of an N-extein sequence followed by a 123-AA intein sequence, whereas the dnaE-c product consists of a 36-AA intein sequence followed by a C-extein sequence.^[11]

See also

• Intragenomic conflict
• Intron
• Protein splicing
• RNA splicing

References

• de Grey, Aubrey D. N. J. (2000). "Mitochondrial gene therapy: an arena for the biomedical use of inteins". Trends Biotechnol 18 (9): 394–399. doi:10.1016/S0167-7799(00)01476-1. ISSN 0167-7799. PMID 10942964. Retrieved 2008-11-30. 

• Gogarten, J Peter; Elena Hilario (2006). "Inteins, introns, and homing endonucleases: recent revelations about the life cycle of parasitic genetic elements". BMC Evol Biol 6: 94. doi:10.1186/1471-2148-6-94. ISSN 1471-2148. PMC 1654191. PMID 17101053. Retrieved 2008-11-30.  Cite uses deprecated parameters (help)

Chong, S., Mersha, F. B., Comb, D. G., Scott, M. E., Landry, D., Vence, L. M., Perler, Francine B, 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), 271-281. doi:10.1016/S0378-1119(97)00105-4

Fong, B. a, & Wood, D. W. (2010). Expression and purification of ELP-intein-tagged target proteins in high cell density E. coli fermentation. Microbial cell factories, 9(1), 77. BioMed Central Ltd. doi:10.1186/1475-2859-9-77

Xing, L., Wu, W., Zhou, B., & Lin, Z. (2011). Streamlined protein expression and purification using cleavable self-aggregating tags. Microbial cell factories, 10(1), 42. BioMed Central Ltd. doi:10.1186/1475-2859-10-42

Perler, F. B. (2002). InBase: the Intein Database. Nucleic Acids Research, 30(1), 383-384. doi:10.1093/nar/30.1.383

External links

• The Intein Database
• Shmuel Pietrokovski's Intein database