Editing Protein splicing

{{Short description|The post-translational removal of peptide sequences from within a protein sequence}}
[[File:intein mech.png|thumb|upright=1.25|alt=mechanism of protein splicing involving inteins|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.]]
'''Protein splicing''' is an intramolecular reaction of a particular [[protein]] in which an internal protein segment (called an '''intein''') is removed from a precursor protein with a ligation of [[C-terminus|C-terminal]] and [[N-terminus|N-terminal]] external proteins (called [[extein]]s) on both sides. The splicing junction of the precursor protein is mainly a [[cysteine]] or a [[serine]], which are [[amino acid]]s containing a [[Nucleophile|nucleophilic]] [[side chain]]. The protein splicing reactions which are known now do not require exogenous cofactors or energy sources such as [[adenosine triphosphate]] (ATP) or [[guanosine triphosphate]] (GTP). Normally, '''splicing''' is associated only with [[Splicing (genetics)|pre-mRNA splicing]]. 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]]''<ref>{{cite journal | last1=Bowman | first1=EJ | last2=Tenney | first2=K | last3=Bowman | first3=BJ | date=Oct 1988 | title=Isolation of genes encoding the Neurospora vacuolar ATPase. Analysis of vma-1 encoding the 67-kDa subunit reveals homology to other ATPases | journal=J Biol Chem | volume=263 | issue=28| pages=13994–4001 | doi=10.1016/S0021-9258(18)68175-X | pmid=2971651 | doi-access=free }}</ref> and carrot<ref>{{cite journal | last1=Zimniak | first1=L | last2=Dittrich | first2=P | last3=Gogarten | first3=JP | last4=Kibak | first4=H | last5=Taiz | first5=L | date=Jul 1988 | title=The cDNA sequence of the 69-kDa subunit of the carrot vacuolar H+-ATPase. Homology to the beta-chain of F0F1-ATPases | journal=J Biol Chem | volume=263 | issue=19| pages=9102–12 | doi=10.1016/S0021-9258(19)76514-4 | pmid=2897965 | doi-access=free }}</ref> [[vacuole|vacuolar]] [[ATPase]] (without intein) and the [[homology (biology)|homologous]] gene in yeast (with intein) that was first described as a putative [[ion transporter|calcium ion transporter]].<ref>{{cite journal | last1=Shih | first1=CK | last2=Wagner | first2=R | last3=Feinstein | first3=S | last4=Kanik-Ennulat | first4=C | last5=Neff | first5=N | date=Aug 1988 | title=A dominant trifluoperazine resistance gene from Saccharomyces cerevisiae has homology with F0F1 ATP synthase and confers calcium-sensitive growth | journal=Mol Cell Biol | volume=8 | issue=8| pages=3094–103 | pmc=363536 | pmid=2905423 | doi=10.1128/mcb.8.8.3094}}</ref> In 1990 Hirata ''et al.''<ref>{{cite journal | last1=Hirata | first1=R | last2=Ohsumk | first2=Y | last3=Nakano | first3=A | last4=Kawasaki | first4=H | last5=Suzuki | first5=K | last6=Anraku | first6=Y | date=Apr 1990 | title=Molecular structure of a gene, VMA1, encoding the catalytic subunit of H(+)-translocating adenosine triphosphatase from vacuolar membranes of Saccharomyces cerevisiae | journal=J Biol Chem | volume=265 | issue=12| pages=6726–33 | doi=10.1016/S0021-9258(19)39210-5 | pmid=2139027 | doi-access=free }}</ref> 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-domain system|three domains of life]] (eukaryotes, bacteria, and archaea) and in [[virus]]es.

Protein splicing was unanticipated and its mechanisms were discovered by two groups (Anraku <ref>{{cite journal |vauthors=Hirata R, Ohsumk Y, Nakano A, Kawasaki H, Suzuki K, Anraku Y |title=Molecular structure of a gene, VMA1, encoding the catalytic subunit of H(+)-translocating adenosine triphosphatase from vacuolar membranes of Saccharomyces cerevisiae |journal=J. Biol. Chem. |volume=265 |issue=12 |pages=6726–33 |date=April 1990 |doi=10.1016/S0021-9258(19)39210-5 |pmid=2139027 |doi-access=free }}</ref> and Stevens<ref>{{cite journal |vauthors=Kane PM, Yamashiro CT, Wolczyk DF, Neff N, Goebl M, Stevens TH |title=Protein splicing converts the yeast TFP1 gene product to the 69-kD subunit of the vacuolar H(+)-adenosine triphosphatase |journal=Science |volume=250 |issue=4981 |pages=651–7 |date=November 1990 |pmid=2146742 |doi= 10.1126/science.2146742|bibcode=1990Sci...250..651K }}</ref>) in 1990. They both discovered a ''[[Saccharomyces cerevisiae]]'' VMA1 in a precursor of a [[vacuolar ATPase|vacuolar H<sup>+</sup>-ATPase]] enzyme. The amino acid sequence of the N- and C-termini corresponded to 70% DNA sequence of that of a vacuolar H<sup>+</sup>-ATPase from other organisms, while the amino acid sequence of the central position corresponded to 30% of the total DNA sequence of the [[yeast]] HO [[nuclease]].

Many [[gene]]s 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 [[Parasite|parasitic]]. According to the gene centered view of evolution, most genes are "selfish" only insofar as to compete with other genes or [[allele]]s 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.<ref name="SwithersSoucy2012">{{cite journal| last1=Swithers| first1=Kristen S.| last2=Soucy| first2=Shannon M.| last3=Gogarten| first3=J. Peter| title=The Role of Reticulate Evolution in Creating Innovation and Complexity| journal=International Journal of Evolutionary Biology| volume=2012| year=2012| pages=1–10| issn=2090-8032| doi=10.1155/2012/418964| pmid=22844638| pmc=3403396| doi-access=free}}</ref><ref>{{cite book | last=Dawkins | first=Richard| author-link=Richard Dawkins | title=[[The Selfish Gene]] | year=1976 | publisher=Oxford University Press}}</ref>

As of December 2019, the UniProtKB database contains 188 entries manually annotated as inteins, ranging from just tens of amino acid residues to thousands.<ref>{{Cite journal |date=2016-11-29 |title=UniProt: the universal protein knowledgebase |url=http://dx.doi.org/10.1093/nar/gkw1099 |journal=Nucleic Acids Research |volume=45 |issue=D1 |pages=D158–D169 |doi=10.1093/nar/gkw1099 |pmid=27899622 |pmc=5210571 |issn=0305-1048}}</ref> 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 protein]]s, 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.<ref name="Perler2002">{{cite journal| last1=Perler| first1=F. B.| title=InBase: the Intein Database| journal=Nucleic Acids Research| volume=30| issue=1| year=2002| pages=383–384| issn=1362-4962| doi=10.1093/nar/30.1.383| pmid=11752343| pmc=99080}}</ref> The abundance of intein in fungi indicates [[Horizontal gene transfer|lateral transfer]] of intein-containing genes. While in eubacteria and archaea, there are 289 and 182 currently known inteins. Not surprisingly, most intein in eubacteria and archaea are found to be inserted into nucleic acid metabolic protein, like fungi.<ref name="Perler2002" />

Inteins vary greatly, but many of the same intein-containing proteins are found in a number of species. For example, pre-mRNA processing factor 8 ([[Prp8]]) protein, instrumental in the [[spliceosome]], has seven different intein insertion sites across eukaryotic species.<ref>{{Cite journal |last1=Green |first1=Cathleen M. |last2=Li |first2=Zhong |last3=Smith |first3=Aaron D. |last4=Novikova |first4=Olga |last5=Bacot-Davis |first5=Valjean R. |last6=Gao |first6=Fengshan |last7=Hu |first7=Saiyang |last8=Banavali |first8=Nilesh K. |last9=Thiele |first9=Dennis J. |last10=Li |first10=Hongmin |last11=Belfort |first11=Marlene |date=2019-10-10 |title=Spliceosomal Prp8 intein at the crossroads of protein and RNA splicing |journal=PLOS Biology |language=en |volume=17 |issue=10 |pages=e3000104 |doi=10.1371/journal.pbio.3000104 |issn=1545-7885 |pmc=6805012 |pmid=31600193 |doi-access=free }}</ref> Intein-containing Prp8 is most commonly found in fungi, but is also seen in [[Amoebozoa]], [[Chlorophyta]], [[Capsaspora]], and [[Choanoflagellida]]. Many mycobacteria contain inteins within [[dnaB helicase|DnaB]] (bacterial replicative helicase), [[RecA]] (bacterial DNA recombinase), and SufB ([[Iron–sulfur cluster biosynthesis|FeS cluster assembly]] protein).<ref name=":0"/><ref name="Wall 2021">{{Cite journal |last1=Wall |first1=Diana A. |last2=Tarrant |first2=Seanan P. |last3=Wang |first3=Chunyu |last4=Mills |first4=Kenneth V. |last5=Lennon |first5=Christopher W. |date=2021 |title=Intein Inhibitors as Novel Antimicrobials: Protein Splicing in Human Pathogens, Screening Methods, and Off-Target Considerations |journal=Frontiers in Molecular Biosciences |volume=8 |page=752824 |doi=10.3389/fmolb.2021.752824 |issn=2296-889X |pmc=8529194 |pmid=34692773 |doi-access=free }}</ref> There is remarkable variety within the structure and number of DnaB inteins, both within the mycobacterium genus and beyond. Interestingly, intein-containing DnaB is also found in the [[chloroplast]]s of algae.<ref>{{Cite journal |last1=Green |first1=Cathleen M. |last2=Novikova |first2=Olga |last3=Belfort |first3=Marlene |date=2018-01-24 |title=The dynamic intein landscape of eukaryotes |journal=Mobile DNA |volume=9 |issue=1 |pages=4 |doi=10.1186/s13100-018-0111-x |issn=1759-8753 |pmc=5784728 |pmid=29416568 |doi-access=free }}</ref> Intein-containing proteins found in archaea include RadA (RecA homolog), RFC, PolB, RNR.<ref name="Novikova 14490–14497">{{Cite journal |last1=Novikova |first1=Olga |last2=Topilina |first2=Natalya |last3=Belfort |first3=Marlene |date=May 2014 |title=Enigmatic Distribution, Evolution, and Function of Inteins |journal=Journal of Biological Chemistry |volume=289 |issue=21 |pages=14490–14497 |doi=10.1074/jbc.r114.548255 |issn=0021-9258 |pmc=4031506 |pmid=24695741 |doi-access=free }}</ref> Many of the same intein-containing proteins (or their homologs) are found in two or even all three domains of life. Inteins are also seen in the proteomes encoded by bacteriophages and eukaryotic viruses. Viruses may have been involved as vectors of intein distribution across the wide variety of intein containing organisms.<ref name="Novikova 14490–14497"/>

== Mechanism ==
{{missing information|section|Conserved A, B, F, G blocks |date=November 2023}}
The process for class 1 inteins 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 [[nucleophile|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-terminus|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]] (Asn), 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 [[amine|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, [[Chemical ligation|ligated]] protein.<ref>{{cite journal | vauthors=Noren CJ, Wang J, Perler FB | title=Dissecting the chemistry of protein splicing and its applications| journal=Angew Chem Int Ed Engl| volume=39| issue=3| pages=450–66| year=2000| pmid=10671234| doi=10.1002/(sici)1521-3773(20000204)39:3<450::aid-anie450>3.3.co;2-6}}</ref>

Class 2 inteins have no nucleophilic first side chain, only an alanine. Instead, the reaction starts directly with a nucleophilic displacement, with the first residue of the C-extein atticking the peptide carboxyl on the final residue of the N-extein. The rest proceeds as usual, starting with Asn turning into a cyclic imide.<ref name="pmid33339089"/>

Class 3 inteins have no nucleophilic first side chain, only an alanine, yet they have an internal noncontiguous "WCT" motif. The internal C (cysteine) residue attacks the peptide carboxyl on the final residue of the N-extein (nucleophilic displacement). Transesterification occurs when the first residue of the C-extein attacks the newly formed thioester. The rest proceeds as usual.<ref name=pmid21317331/>

The mechanism for the splicing effect is a naturally occurring analogy to the technique for chemically generating medium-sized proteins called [[native chemical ligation]].

==Intein==
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]]  during protein splicing.<ref>{{cite journal |author1=Anraku, Y |author2=Mizutani, R |author3=Satow, Y  | title=Protein splicing: its discovery and structural insight into novel chemical mechanisms | journal=IUBMB Life | volume=57 | issue=8 | pages=563–74 | year=2005 | pmid=16118114 | doi=10.1080/15216540500215499| doi-access=free }}</ref> Inteins have also been called '''protein introns''', by analogy with (RNA) [[intron]]s. 
[[File:Intein_splicing_dogma.png|thumb|248x248px|Intein splicing occurs post-translationally in a self-catalytic process. Here, the extein is shown in red and the intein in blue. Image created with Biorender.com.]]

===Naming conventions===
The first part of an intein name is based on the [[Binomial nomenclature|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 [[Directionality (molecular biology)|5{{prime}} to 3{{prime}}]] 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'').  A different disambiguating convention is to place a lowercase "i" after the source protein name, ''e.g.'' "Msm DnaBi1".<ref>{{cite journal |last1=Kelley |first1=Danielle S. |last2=Lennon |first2=Christopher W. |last3=Li |first3=Zhong |last4=Miller |first4=Michael R. |last5=Banavali |first5=Nilesh K. |last6=Li |first6=Hongmin |last7=Belfort |first7=Marlene |title=Mycobacterial DnaB helicase intein as oxidative stress sensor |journal=Nature Communications |date=19 October 2018 |volume=9 |issue=1 |page=4363 |doi=10.1038/s41467-018-06554-x|pmid=30341292 |pmc=6195587 |bibcode=2018NatCo...9.4363K |doi-access=free }}</ref>

===Types of inteins===
Inteins can be classified on many criteria.
* Based on how they splice themselves out, they can be classified into ''cis-splicing'' (which means that they splice themselves out) or ''trans-splicing'' (which means they need outside help). Most studied inteins are cis-splicing. Split inteins (see below) usually involves two halves helping each other out, so they are ''trans-splicing''.<ref name="pmid33339089">{{cite journal |last1=Nanda |first1=A |last2=Nasker |first2=SS |last3=Mehra |first3=A |last4=Panda |first4=S |last5=Nayak |first5=S |title=Inteins in Science: Evolution to Application. |journal=Microorganisms |date=16 December 2020 |volume=8 |issue=12 |page=2004 |doi=10.3390/microorganisms8122004 |pmid=33339089 |pmc=7765530 |doi-access=free }}</ref>
* Based on whether they contain the endonuclease domain. Ones that have an endonuclease domain is called a "maxi-intein", otherwise a "mini-intein".<ref name="pmid33339089"/>
* Based on their splicing mechanism, which can be partially<ref name=pmid21317331/> inferred based on the sequence. Class 1 intein is the most-studied type and are marked by a cysteine or serine as the first residue. Class 2 intein, or "alanine intein" has alanine as the first residue and no WCT motif. Class 3 intein has alanine as the first residue and a noncontiguous "WCT" motif.<ref name="pmid33339089"/> It has also been proposed that inteins that start with a serine and contain a "WCT" motif should be also classed as class 3.<ref name=pmid21317331>{{cite journal |last1=Tori |first1=K |last2=Perler |first2=FB |title=Expanding the definition of class 3 inteins and their proposed phage origin. |journal=Journal of Bacteriology |date=April 2011 |volume=193 |issue=8 |pages=2035–41 |doi=10.1128/JB.01407-10 |pmid=21317331|pmc=3133030 |doi-access=free }}</ref>

==== Full and mini inteins ====
Inteins can contain a [[Homing endonuclease| 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 repair#Double-strand breaks|DNA double-stranded break repair]] (DSBR) system, which then repairs the break, thus copying the intein-coding DNA into a previously intein-free site.<ref name="pmid33339089"/> 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| date=November 2011}}

==== 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 holoenzyme|DNA polymerase III]], is encoded by two separate genes, ''dnaE-n'' and ''dnaE-c''. The ''dnaE-n'' [[gene product|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.<ref name="DnaE">{{Cite journal | doi=10.1073/pnas.95.16.9226 | last1=Wu | first1=H. | last2=Hu | first2=Z. | last3=Liu | first3=X. Q. 
| title=Protein trans-splicing by a split intein encoded in a split DnaE gene of Synechocystis sp. PCC6803 
| journal=Proceedings of the National Academy of Sciences of the United States of America | volume=95 | issue=16 | pages=9226–9231 | year=1998 | pmid=9689062 | pmc=21320| bibcode=1998PNAS...95.9226W | doi-access=free }}</ref>

==Applications 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&ndash;800 [[Amino acid|AA]]s. Inteins have been engineered for particular applications such as [[peptide synthesis|protein semisynthesis]]<ref>{{cite journal | vauthors=Schwarzer D, Cole PA | title=Protein semisynthesis and expressed protein ligation: chasing a protein's tail | journal=Curr Opin Chem Biol | volume=9 | issue=6 | pages=561–9 | year=2005 | pmid=16226484 | doi=10.1016/j.cbpa.2005.09.018}}</ref> and the selective labeling of protein segments, which is useful for [[Protein NMR|NMR]] studies of large proteins.<ref>{{cite journal | vauthors=Muralidharan V, Muir TW | title=Protein ligation: an enabling technology for the biophysical analysis of proteins | journal=Nat. Methods | volume=3 | issue=6 | pages=429–38 | year=2006 | pmid=16721376 | doi=10.1038/nmeth886| s2cid=12550693 }}</ref>

[[Enzyme inhibitor|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 [[hydrophobe|hydrophobic]] proteins normally encoded by the [[mitochondria]]l genome, for example in [[gene therapy]].<ref name="de Grey2000">{{cite journal| last1=de Grey| first1=Aubrey D.N.J| title=Mitochondrial gene therapy: an arena for the biomedical use of inteins| journal=Trends in Biotechnology| volume=18| issue=9| year=2000| pages=394–399| issn=0167-7799| doi=10.1016/S0167-7799(00)01476-1| pmid=10942964}}</ref> 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]].

Affinity tags have been widely used to purify recombinant proteins, as they allow the accumulation of recombinant protein with little impurities. However, the affinity tag must be removed by proteases in the final purification step. The extra proteolysis step raises the problems of protease specificity in removing affinity tags from recombinant protein, and the removal of the digestion product. This problem can be avoided by fusing an affinity tag to self-cleavable inteins in a controlled environment. The first generation of expression vectors of this kind used modified ''Saccharomyces cerevisiae'' VMA (Sce VMA) intein. Chong et al.<ref name="ChongMersha1997">{{cite journal| last1=Chong| first1=Shaorong| last2=Mersha| first2=Fana B| last3=Comb| first3=Donald G| last4=Scott| first4=Melissa E| last5=Landry| first5=David| last6=Vence| first6=Luis M| last7=Perler| first7=Francine B| last8=Benner| first8=Jack| last9=Kucera| first9=Rebecca B| last10=Hirvonen| first10=Christine A| last11=Pelletier| first11=John J| last12=Paulus| first12=Henry| last13=Xu| first13=Ming-Qun| title=Single-column purification of free recombinant proteins using a self-cleavable affinity tag derived from a protein splicing element| journal=Gene| volume=192| issue=2| year=1997| pages=271–281| issn=0378-1119| doi=10.1016/S0378-1119(97)00105-4| pmid=9224900}}</ref> used a chitin binding domain (CBD) from ''[[Bacillus circulans]]'' as an affinity tag, and fused this tag with a modified Sce VMA intein. The modified intein undergoes a self-cleavage reaction at its N-terminal peptide linkage with [[1,4-dithiothreitol]] (DTT), [[β-mercaptoethanol]] (β-ME), or [[cystine]] at low temperatures over a broad pH range. After expressing the recombinant protein, the cell homogenate is passed through the column containing [[chitin]]. This allows the CBD of the chimeric protein to bind to the column. Furthermore, when the temperature is lowered and the molecules described above pass through the column, the chimeric protein undergoes self-splicing and only the target protein is eluted. This novel technique eliminates the need for a proteolysis step, and modified Sce VMA stays in column attached to chitin through CBD.<ref name="ChongMersha1997" />

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.<ref name="FongWood2010">{{cite journal| last1=Fong| first1=Baley A| last2=Wood| first2=David W| title=Expression and purification of ELP-intein-tagged target proteins in high cell density E. coli fermentation| journal=Microbial Cell Factories| volume=9| issue=1| year=2010| pages=77| issn=1475-2859| doi=10.1186/1475-2859-9-77| pmc=2978133| pmid=20959011| doi-access=free}}</ref> 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.<ref name="FongWood2010" /> 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.<ref name="XingWu2011">{{cite journal| last1=Xing| first1=Lei| last2=Wu| first2=Wei| last3=Zhou| first3=Bihong| last4=Lin| first4=Zhanglin| title=Streamlined protein expression and purification using cleavable self-aggregating tags| journal=Microbial Cell Factories| volume=10| issue=1| year=2011| pages=42| issn=1475-2859| doi=10.1186/1475-2859-10-42| pmc=3124420| pmid=21631955| doi-access=free}}</ref>

== Applications in Antimicrobial Development ==
Over the last twenty years, there has been increasing interest in leveraging inteins for [[antimicrobial]] applications.<ref name=":0">{{Cite journal |last1=Tharappel |first1=Anil Mathew |last2=Li |first2=Zhong |last3=Li |first3=Hongmin |date=2022 |title=Inteins as Drug Targets and Therapeutic Tools |journal=Frontiers in Molecular Biosciences |volume=9 |page=821146 |doi=10.3389/fmolb.2022.821146 |issn=2296-889X |pmc=8861304 |pmid=35211511 |doi-access=free }}</ref> Intein splicing is found exclusively in unicellular organisms, with a particularly high abundance in pathogenic microorganisms.<ref>{{Cite journal |last1=Shah |first1=Neel H. |last2=Muir |first2=Tom W. |date=2013-12-24 |title=Inteins: nature's gift to protein chemists |journal=Chemical Science |language=en |volume=5 |issue=2 |pages=446–461 |doi=10.1039/C3SC52951G |issn=2041-6539 |pmc=3949740 |pmid=24634716}}</ref> Furthermore, inteins are commonly found within housekeeping proteins and/or proteins involved in the survival of the organism within a human host. Post-translational intein removal is necessary for the protein to properly fold and function. For example, Gaëlle Huet ''et al.'' demonstrated that in ''[[Mycobacterium tuberculosis]]'', unspliced SufB prevents the formation of the SufBCD complex, a component of the SUF machinery.<ref>{{Cite journal |last1=Huet |first1=Gaëlle |last2=Castaing |first2=Jean-Philippe |last3=Fournier |first3=Didier |last4=Daffé |first4=Mamadou |last5=Saves |first5=Isabelle |date=May 2006 |title=Protein Splicing of SufB Is Crucial for the Functionality of the Mycobacterium tuberculosis SUF Machinery |journal=Journal of Bacteriology |language=en |volume=188 |issue=9 |pages=3412–3414 |doi=10.1128/JB.188.9.3412-3414.2006 |issn=0021-9193 |pmc=1447444 |pmid=16621837}}</ref> As such, the inhibition of intein splicing may serve as a powerful platform for the development of antimicrobials.

Current research on intein splicing inhibitors has focused on developing [[antimycobacterial]]s (''M. tb.'' has three intein-containing proteins), as well as agents active against pathogenic fungi ''[[Cryptococcus]]'' and ''Aspergillus.''<ref name="Wall 2021"/> Cisplatin and similar platinum-containing compounds inhibit splicing of the ''M. tb.'' RecA intein through coordinating to catalytic residues.<ref>{{Cite journal |last1=Chan |first1=Hon |last2=Pearson |first2=C. Seth |last3=Green |first3=Cathleen M. |last4=Li |first4=Zhong |last5=Zhang |first5=Jing |last6=Belfort |first6=Georges |last7=Shekhtman |first7=Alex |last8=Li |first8=Hongmin |last9=Belfort |first9=Marlene |date=October 2016 |title=Exploring Intein Inhibition by Platinum Compounds as an Antimicrobial Strategy |journal=Journal of Biological Chemistry |volume=291 |issue=43 |pages=22661–22670 |doi=10.1074/jbc.m116.747824 |issn=0021-9258 |pmc=5077202 |pmid=27609519 |doi-access=free }}</ref> Divalent cations, such as copper (II) and zinc (II) ions, function similarly to reversibly inhibit splicing.<ref name=":0" /> However, neither of these methods are currently suitable for an effective and safe antibiotic. The fungal Prp8 intein is also inhibited by divalent cations and cisplatin through interfering with the catalytic Cys1 residue.<ref name=":0"/> In 2021, Li ''et al.'' showed that small molecule inhibitors of Prp8 intein splicing were selective and effective at slowing the growth of [[Cryptococcus neoformans|''C. neoformans'']] and [[Cryptococcus gattii|''C. gattii'']], providing exciting evidence for the antimicrobial potential of intein splicing inhibitors.<ref>{{Cite journal |last1=Li |first1=Zhong |last2=Tharappel |first2=Anil Mathew |last3=Xu |first3=Jimin |last4=Lang |first4=Yuekun |last5=Green |first5=Cathleen M. |last6=Zhang |first6=Jing |last7=Lin |first7=Qishan |last8=Chaturvedi |first8=Sudha |last9=Zhou |first9=Jia |last10=Belfort |first10=Marlene |last11=Li |first11=Hongmin |date=2021-01-12 |title=Small-molecule inhibitors for the Prp8 intein as antifungal agents |journal=Proceedings of the National Academy of Sciences |language=en |volume=118 |issue=2 |pages=e2008815118 |doi=10.1073/pnas.2008815118 |issn=0027-8424 |pmc=7812778 |pmid=33397721 |bibcode=2021PNAS..11808815L |doi-access=free }}</ref>

== See also ==
* [[Intragenomic conflict]]
* [[Intron]]
* [[Protein tag]]
* [[RNA splicing]]

==References==
{{reflist}}

==Further reading==
*{{Cite journal| doi=10.1186/1471-2148-6-94| pmid=17101053| pmc=1654191| issn=1471-2148| volume=6| pages=94<!-- BMC papers have article instead of page numbers -->| last=Gogarten| first=J Peter| author2=Elena Hilario| title=Inteins, introns, and homing endonucleases: recent revelations about the life cycle of parasitic genetic elements| journal=BMC Evol Biol| year=2006| issue=1| doi-access=free}}

==External links==
*[http://www.inteins.com/ The Intein Database]
*[https://web.archive.org/web/20041204100727/http://bioinformatics.weizmann.ac.il/~pietro/inteins/ Shmuel Pietrokovski's Intein database]
*[https://web.archive.org/web/20071203144533/http://starokadomskyy.narod.ru/work_intein_eng.html Short review]
*[https://web.archive.org/web/20110827215956/http://starokadomskyy.narod.ru/Protein_splicing_eng.pdf Starokadomskyy PL. Protein Splicing, 2007]
*[https://web.archive.org/web/20100802024011/http://www.neb.com/neb/inteins.html Protein splicing mechanism and intein structure]
* {{MeshName|Protein+Splicing}}

[[Category:Proteins]]
[[Category:Protein biosynthesis]]