Efficient and robust preparation of tyrosine phosphorylated intrinsically disordered proteins.
Biotechniques
; 67(1): 16-22, 2019 07.
Article
em En
| MEDLINE
| ID: mdl-31092000
ABSTRACT
Intrinsically disordered proteins (IDPs) are subject to post-translational modifications. This allows the same polypeptide to be involved in different interaction networks with different consequences, ranging from regulatory signalling networks to the formation of membrane-less organelles. We report a robust method for co-expression of modification enzyme and SUMO-tagged IDPs with a subsequent purification procedure that allows for the production of modified IDP. The robustness of our protocol is demonstrated using a challenging system RNA polymerase II C-terminal domain (CTD); that is, a low-complexity repetitive region with multiple phosphorylation sites. In vitro phosphorylation approaches fail to yield multiple-site phosphorylated CTD, whereas our in vivo protocol allows the rapid production of near homogeneous phosphorylated CTD at a low cost. These samples can be used in functional and structural studies.
Palavras-chave
Texto completo:
1
Coleções:
01-internacional
Temas:
Geral
Base de dados:
MEDLINE
Assunto principal:
Tirosina
/
Proteínas Intrinsicamente Desordenadas
Limite:
Humans
Idioma:
En
Revista:
Biotechniques
Ano de publicação:
2019
Tipo de documento:
Article
País de afiliação:
República Tcheca