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Inteins, valuable genetic elements in molecular biology and biotechnology.

Elleuche S, Pöggeler S - Appl. Microbiol. Biotechnol. (2010)

Bottom Line: They are found in organisms in all three domains of life, and in viral proteins.Intein excision is a posttranslational process that does not require auxiliary enzymes or cofactors.This review summarizes the catalytic activities and structures of inteins, and focuses on the advantages of some recent intein applications in molecular biology and biotechnology.

View Article: PubMed Central - PubMed

Affiliation: Institute of Technical Microbiology, Technical University Hamburg-Harburg, Kasernenstr. 12, 21073, Hamburg, Germany.

ABSTRACT
Inteins are internal protein elements that self-excise from their host protein and catalyze ligation of the flanking sequences (exteins) with a peptide bond. They are found in organisms in all three domains of life, and in viral proteins. Intein excision is a posttranslational process that does not require auxiliary enzymes or cofactors. This self-excision process is called protein splicing, by analogy to the splicing of RNA introns from pre-mRNA. Protein splicing involves only four intramolecular reactions, and a small number of key catalytic residues in the intein and exteins. Protein-splicing can also occur in trans. In this case, the intein is separated into N- and C-terminal domains, which are synthesized as separate components, each joined to an extein. The intein domains reassemble and link the joined exteins into a single functional protein. Understanding the cis- and trans-protein splicing mechanisms led to the development of intein-mediated protein-engineering applications, such as protein purification, ligation, cyclization, and selenoprotein production. This review summarizes the catalytic activities and structures of inteins, and focuses on the advantages of some recent intein applications in molecular biology and biotechnology.

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Self-circularization of peptides. a Self-circularization of proteins using the TWIN-system. A target protein is embedded between two intein sequences, which are modified for N- or C-terminal cleavage, respectively. The inducible splicing reaction of the inteins leads to the generation of an activated thioester residue and an N-terminal Cys for the spontaneous circularization of the linear peptide. b Utilization of the Split Intein-mediated Ciruclar Ligation Of Peptides a ProteinS (SICLOPPS) also enables the circularization of peptides. In this system, the order of the naturally split Synechocystis sp. Ssp DnaE intein is inverted (IC–target protein–IN), and the reconstitution of the Ssp DnaE intein allows the efficient cyclization of the target protein
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Fig5: Self-circularization of peptides. a Self-circularization of proteins using the TWIN-system. A target protein is embedded between two intein sequences, which are modified for N- or C-terminal cleavage, respectively. The inducible splicing reaction of the inteins leads to the generation of an activated thioester residue and an N-terminal Cys for the spontaneous circularization of the linear peptide. b Utilization of the Split Intein-mediated Ciruclar Ligation Of Peptides a ProteinS (SICLOPPS) also enables the circularization of peptides. In this system, the order of the naturally split Synechocystis sp. Ssp DnaE intein is inverted (IC–target protein–IN), and the reconstitution of the Ssp DnaE intein allows the efficient cyclization of the target protein

Mentions: The pTWIN vector of the TWo INtein system contains two engineered inteins (Evans and Xu 1999). The mutated Synechocystis sp. Ssp DnaB intein allows C-terminal cleavage, while Mycobacterium xenopy Mxe GyrA intein undergoes N-terminal cleavage. The combination of both proteins fused to the N- and C-terminal ends of a target protein enables the production of an N-terminal Cys residue and an activated thioester at the C-terminus, which react, resulting in cyclization (Fig. 5a). A disadvantage to this method is the low cleavage efficiency of the Ssp DnaB intein, which is influenced by the second and third amino acid residues following the required Cys at the N-terminus of the target protein. The introduction of a non-native linker sequence improves cleavage efficiency, but also has the potential to interfere with the biological activity of the cyclic protein. Another problem is the possibility of polymerization instead of cyclization by activated peptides (Xu and Evans 2001).Fig. 5


Inteins, valuable genetic elements in molecular biology and biotechnology.

Elleuche S, Pöggeler S - Appl. Microbiol. Biotechnol. (2010)

Self-circularization of peptides. a Self-circularization of proteins using the TWIN-system. A target protein is embedded between two intein sequences, which are modified for N- or C-terminal cleavage, respectively. The inducible splicing reaction of the inteins leads to the generation of an activated thioester residue and an N-terminal Cys for the spontaneous circularization of the linear peptide. b Utilization of the Split Intein-mediated Ciruclar Ligation Of Peptides a ProteinS (SICLOPPS) also enables the circularization of peptides. In this system, the order of the naturally split Synechocystis sp. Ssp DnaE intein is inverted (IC–target protein–IN), and the reconstitution of the Ssp DnaE intein allows the efficient cyclization of the target protein
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2874743&req=5

Fig5: Self-circularization of peptides. a Self-circularization of proteins using the TWIN-system. A target protein is embedded between two intein sequences, which are modified for N- or C-terminal cleavage, respectively. The inducible splicing reaction of the inteins leads to the generation of an activated thioester residue and an N-terminal Cys for the spontaneous circularization of the linear peptide. b Utilization of the Split Intein-mediated Ciruclar Ligation Of Peptides a ProteinS (SICLOPPS) also enables the circularization of peptides. In this system, the order of the naturally split Synechocystis sp. Ssp DnaE intein is inverted (IC–target protein–IN), and the reconstitution of the Ssp DnaE intein allows the efficient cyclization of the target protein
Mentions: The pTWIN vector of the TWo INtein system contains two engineered inteins (Evans and Xu 1999). The mutated Synechocystis sp. Ssp DnaB intein allows C-terminal cleavage, while Mycobacterium xenopy Mxe GyrA intein undergoes N-terminal cleavage. The combination of both proteins fused to the N- and C-terminal ends of a target protein enables the production of an N-terminal Cys residue and an activated thioester at the C-terminus, which react, resulting in cyclization (Fig. 5a). A disadvantage to this method is the low cleavage efficiency of the Ssp DnaB intein, which is influenced by the second and third amino acid residues following the required Cys at the N-terminus of the target protein. The introduction of a non-native linker sequence improves cleavage efficiency, but also has the potential to interfere with the biological activity of the cyclic protein. Another problem is the possibility of polymerization instead of cyclization by activated peptides (Xu and Evans 2001).Fig. 5

Bottom Line: They are found in organisms in all three domains of life, and in viral proteins.Intein excision is a posttranslational process that does not require auxiliary enzymes or cofactors.This review summarizes the catalytic activities and structures of inteins, and focuses on the advantages of some recent intein applications in molecular biology and biotechnology.

View Article: PubMed Central - PubMed

Affiliation: Institute of Technical Microbiology, Technical University Hamburg-Harburg, Kasernenstr. 12, 21073, Hamburg, Germany.

ABSTRACT
Inteins are internal protein elements that self-excise from their host protein and catalyze ligation of the flanking sequences (exteins) with a peptide bond. They are found in organisms in all three domains of life, and in viral proteins. Intein excision is a posttranslational process that does not require auxiliary enzymes or cofactors. This self-excision process is called protein splicing, by analogy to the splicing of RNA introns from pre-mRNA. Protein splicing involves only four intramolecular reactions, and a small number of key catalytic residues in the intein and exteins. Protein-splicing can also occur in trans. In this case, the intein is separated into N- and C-terminal domains, which are synthesized as separate components, each joined to an extein. The intein domains reassemble and link the joined exteins into a single functional protein. Understanding the cis- and trans-protein splicing mechanisms led to the development of intein-mediated protein-engineering applications, such as protein purification, ligation, cyclization, and selenoprotein production. This review summarizes the catalytic activities and structures of inteins, and focuses on the advantages of some recent intein applications in molecular biology and biotechnology.

Show MeSH
Related in: MedlinePlus