Domain selective labeling for NMR studies of multidomain proteins by domain ligation using highly active sortase A.
Biochim Biophys Acta Gen Subj
; 1864(2): 129419, 2020 02.
Article
en En
| MEDLINE
| ID: mdl-31449838
ABSTRACT
Structural study of multidomain proteins using NMR is an emerging issue for understanding biological functions. To this end, domain-specific labeling is expected to be a key technology for facilitating the NMR-assignment process and for collecting distance information via spin labeling. To obtain domain-specific labeled samples, use of sortase A as a protein ligation tool is a viable approach. Sortase A enables ligation of separately expressed proteins (domains) through the Leu-Pro-X-Thr-Gly linker. However, the ligation reaction mediated by sortase A is not efficient. Poor yield and long reaction times hamper large-scale preparation using sortase A. Here we report the application of highly active sortases to NMR analyses. Optimal yields can be achieved within several hours when the ligation reaction are mediated by highly active sortases at 4⯰C. We propose that this protocol can contribute to structural analyses of multidomain proteins by NMR.
Palabras clave
Texto completo:
1
Colección:
01-internacional
Base de datos:
MEDLINE
Asunto principal:
Staphylococcus aureus
/
Proteínas Bacterianas
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Cisteína Endopeptidasas
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Aminoaciltransferasas
Idioma:
En
Revista:
Biochim Biophys Acta Gen Subj
Año:
2020
Tipo del documento:
Article
País de afiliación:
Japón