In vitro sortagging of an antibody fab fragment: overcoming unproductive reactions of sortase with water and lysine side chains.
Chembiochem
; 12(11): 1774-80, 2011 Jul 25.
Article
en En
| MEDLINE
| ID: mdl-21656631
ABSTRACT
Sortase A from Staphylococcus aureus attracts growing interest for its use in biotechnological protein modification. This enzyme binds to a short signal sequence at the C terminus of a target protein, cleaves it by formation of an acyl-enzyme intermediate, and subsequently attaches an oligoglycine with a peptide bond. In this work, we explored its usability for the modification of the L19 Fab fragment (specific for fibronectin ED-B), a promising candidate for antibody-based cancer therapy. The Fab fragment was expressed with a sortase signal sequence attached to its light chain, and was successfully modified with a fluorescent oligoglycine probe in good yield. Our interest focused on performance under conditions of limited oligoglycine concentrations. Two unproductive side reactions of sortase were observed. The first was hydrolysis of the acyl-enzyme intermediate; in the second, sortase accepted the ε-amino group of lysine as substrate, thereby resulting in polypeptide crosslinking. In case of the L19 Fab fragment, it led to the covalent connection of the heavy and light chains. Both side reactions were effectively suppressed by sufficient concentrations of the oligoglycine probe.
Texto completo:
1
Colección:
01-internacional
Base de datos:
MEDLINE
Asunto principal:
Proteínas Bacterianas
/
Fragmentos Fab de Inmunoglobulinas
/
Cisteína Endopeptidasas
/
Agua
/
Aminoaciltransferasas
/
Lisina
Límite:
Humans
Idioma:
En
Revista:
Chembiochem
Asunto de la revista:
BIOQUIMICA
Año:
2011
Tipo del documento:
Article
País de afiliación:
Alemania