Editing Protein splicing (section)

==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>