Limits...
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.

Show MeSH

Related in: MedlinePlus

Splicing mechanism of inteins. Intein splicing takes place in four reaction steps. See text for details
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2874743&req=5

Fig3: Splicing mechanism of inteins. Intein splicing takes place in four reaction steps. See text for details

Mentions: Protein splicing is a rapid process of four nucleophilic attacks, mediated by three of the four conserved splice junction residues. In step 1, the splicing process begins with an N−O shift if the first intein residue is Ser, or N−S acyl shift, if the first intein residue is Cys. This forms a (thio)ester bond at the N-extein/intein junction. In step 2, the (thio)ester bond is attacked by the OH- or SH-group of the first residue in the C-extein (Cys, Ser, or Thr). This leads to a transesterification, which transfers the N-extein to the side-chain of the first residue of the C-extein. In step 3, the cyclization of the conserved Asn residue at the C-terminus of the intein releases the intein and links the exteins by a (thio)ester bond. Finally, step 4 is a rearrangement of the (thio)ester bond to a peptide bond by a spontaneous S−N or O−N acyl shift (Fig. 3). Details of the chemical process involved in protein splicing of standard (class I inteins) have been comprehensively described and reviewed (Gogarten et al. 2002; Liu 2000; Noren et al. 2000; Paulus 2000; Saleh and Perler 2006; Starokadomskyy 2007; Tori et al. 2010). In addition to the standard protein splicing pathway, new classes of inteins performing an alternative splicing process have been described recently. These inteins lack the N-terminal Ser or Cys residue and are classified as class 2 and class 3 inteins (Southworth et al. 2000; Tori et al. 2010). Both cannot perform the acyl shift that initiates the splicing reaction in class 1 inteins. In class 2 inteins, present in archaeal KlbA proteins, the first residue of the C-extein (nucleophile Cys) directly attacks the amide bond at the N-terminal splice site junction to form a standard branched intermediate (Johnson et al. 2007; Southworth et al. 2000). In the class 3 intein of the mycobacteriophage Bethlehem DnaB protein, a Cys residue of the conserved block F attacks the peptide bond at the N-terminal splice site junction, forming a branched intermediate with a labile thioester linkage. The N-extein is then transferred by a transesterification to the first residue of the C-extein (Thr), which results in the formation of a standard branched intermediate as in class 1 inteins (Tori et al. 2010).Fig. 3


Inteins, valuable genetic elements in molecular biology and biotechnology.

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

Splicing mechanism of inteins. Intein splicing takes place in four reaction steps. See text for details
© Copyright Policy
Related In: Results  -  Collection

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

Fig3: Splicing mechanism of inteins. Intein splicing takes place in four reaction steps. See text for details
Mentions: Protein splicing is a rapid process of four nucleophilic attacks, mediated by three of the four conserved splice junction residues. In step 1, the splicing process begins with an N−O shift if the first intein residue is Ser, or N−S acyl shift, if the first intein residue is Cys. This forms a (thio)ester bond at the N-extein/intein junction. In step 2, the (thio)ester bond is attacked by the OH- or SH-group of the first residue in the C-extein (Cys, Ser, or Thr). This leads to a transesterification, which transfers the N-extein to the side-chain of the first residue of the C-extein. In step 3, the cyclization of the conserved Asn residue at the C-terminus of the intein releases the intein and links the exteins by a (thio)ester bond. Finally, step 4 is a rearrangement of the (thio)ester bond to a peptide bond by a spontaneous S−N or O−N acyl shift (Fig. 3). Details of the chemical process involved in protein splicing of standard (class I inteins) have been comprehensively described and reviewed (Gogarten et al. 2002; Liu 2000; Noren et al. 2000; Paulus 2000; Saleh and Perler 2006; Starokadomskyy 2007; Tori et al. 2010). In addition to the standard protein splicing pathway, new classes of inteins performing an alternative splicing process have been described recently. These inteins lack the N-terminal Ser or Cys residue and are classified as class 2 and class 3 inteins (Southworth et al. 2000; Tori et al. 2010). Both cannot perform the acyl shift that initiates the splicing reaction in class 1 inteins. In class 2 inteins, present in archaeal KlbA proteins, the first residue of the C-extein (nucleophile Cys) directly attacks the amide bond at the N-terminal splice site junction to form a standard branched intermediate (Johnson et al. 2007; Southworth et al. 2000). In the class 3 intein of the mycobacteriophage Bethlehem DnaB protein, a Cys residue of the conserved block F attacks the peptide bond at the N-terminal splice site junction, forming a branched intermediate with a labile thioester linkage. The N-extein is then transferred by a transesterification to the first residue of the C-extein (Thr), which results in the formation of a standard branched intermediate as in class 1 inteins (Tori et al. 2010).Fig. 3

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