Trypsiligase-Catalyzed Peptide and Protein Ligation.

Catalysis Hydrolysis Ligases / chemistry Models, Molecular Peptides / chemistry Protein Conformation Protein Processing, Post-Translational Proteins / chemistry Recombinant Proteins Substrate Specificity Trypsin / chemistry
Peptide ligation Protein modification Substrate mimetic Substrate-assisted catalysis Transpeptidation Trypsiligase Trypsin variant

Journal

Methods in molecular biology (Clifton, N.J.)
ISSN: 1940-6029
Titre abrégé: Methods Mol Biol
Pays: United States
ID NLM: 9214969

Informations de publication

Date de publication:
2019
Historique:
entrez: 5 6 2019
pubmed: 5 6 2019
medline: 19 3 2020
Statut: ppublish

Résumé

Site-specific incorporation of nonproteinogenic functionalities into protein targets is an important tool in both basic and applied research and represents a major challenge to protein chemists. Chemical labeling methods often target multiple positions within a protein and therefore suffer from a lack of specificity. Enzymatic protein modification is an attractive alternative due to the inherent regioselectivity and stereoselectivity of enzymes. In this chapter we describe the application of the highly specific trypsin variant trypsiligase for the site-specific modification of virtual any target protein. We present two general routes of modification resulting in either N- or C-terminal functionalized protein products. Reactions rapidly proceed under mild conditions and result in homogeneously modified proteins bearing the artificial functionality exclusively at the desired position. We detail protocols for the expression and purification of trypsiligase as well as the synthesis of peptide (ester) substrates. In addition, we provide instructions for the bioconjugation reactions and for the qualitative and quantitative analysis of reaction progress and efficiency.

Identifiants

DOI: 10.1007/978-1-4939-9546-2_7 PMID: 31161506
pubmed: 31161506
doi: 10.1007/978-1-4939-9546-2_7
doi:

Substances chimiques

Peptides 0
Proteins 0
Recombinant Proteins 0
Trypsin EC 3.4.21.4
Ligases EC 6.-

Types de publication

Journal Article

Langues

eng

Sous-ensembles de citation

IM

Pagination

111-133

Références

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Auteurs

Sandra Liebscher (S)

Institute of Biochemistry/Biotechnology, Charles-Tanford-Protein Center, Martin-Luther-University Halle-Wittenberg, Halle, Germany.

Frank Bordusa (F)

Institute of Biochemistry/Biotechnology, Charles-Tanford-Protein Center, Martin-Luther-University Halle-Wittenberg, Halle, Germany. frank.bordusa@biochemtech.uni-halle.de.

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Classifications MeSH

questionsmedicales.fr › Phénomènes chimiques › Catalyse › Trypsiligase-Catalyzed Peptide and Protein Ligation.
questionsmedicales.fr › Phénomènes chimiques › Hydrolyse › Trypsiligase-Catalyzed Peptide and Protein Ligation.
questionsmedicales.fr › Enzymes et coenzymes › Enzymes › Ligases › Trypsiligase-Catalyzed Peptide and Protein Ligation.
questionsmedicales.fr › Techniques d'investigation › Modèles théoriques › Modèles moléculaires › Trypsiligase-Catalyzed Peptide and Protein Ligation.
questionsmedicales.fr › Acides aminés, peptides et protéines › Peptides › Trypsiligase-Catalyzed Peptide and Protein Ligation.
questionsmedicales.fr › Phénomènes chimiques › Phénomènes biochimiques › Conformation moléculaire › Conformation des protéines › Trypsiligase-Catalyzed Peptide and Protein Ligation.
questionsmedicales.fr › Phénomènes génétiques › Régulation de l'expression des gènes › Modification traductionnelle des protéines › Maturation post-traductionnelle des protéines › Trypsiligase-Catalyzed Peptide and Protein Ligation.
questionsmedicales.fr › Acides aminés, peptides et protéines › Protéines › Trypsiligase-Catalyzed Peptide and Protein Ligation.
questionsmedicales.fr › Acides aminés, peptides et protéines › Protéines › Protéines recombinantes › Trypsiligase-Catalyzed Peptide and Protein Ligation.
questionsmedicales.fr › Phénomènes chimiques › Phénomènes biochimiques › Spécificité du substrat › Trypsiligase-Catalyzed Peptide and Protein Ligation.
questionsmedicales.fr › Enzymes et coenzymes › Enzymes › Hydrolases › Peptide hydrolases › Protéases à sérine › Serine endopeptidases › Trypsine › Trypsiligase-Catalyzed Peptide and Protein Ligation.