Adapting recombinant bacterial alkaline phosphatase for nucleotide exchange of small GTPases.
Protein Expr Purif
; 218: 106446, 2024 Jun.
Article
em En
| MEDLINE
| ID: mdl-38395209
ABSTRACT
The small GTPase Rat sarcoma virus proteins (RAS) are key regulators of cell growth and involved in 20-30% of cancers. RAS switches between its active state and inactive state via exchange of GTP (active) and GDP (inactive). Therefore, to study active protein, it needs to undergo nucleotide exchange to a non-hydrolysable GTP analog. Calf intestine alkaline phosphatase bound to agarose beads (CIP-agarose) is regularly used in a nucleotide exchange protocol to replace GDP with a non-hydrolysable analog. Due to pandemic supply problems and product shortages, we found the need for an alternative to this commercially available product. Here we describe how we generated a bacterial alkaline phosphatase (BAP) with an affinity tag bound to an agarose bead. This BAP completely exchanges the nucleotide in our samples, thereby demonstrating an alternative to the commercially available product using generally available laboratory equipment.
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Base de dados:
MEDLINE
Assunto principal:
Proteínas Monoméricas de Ligação ao GTP
Idioma:
En
Ano de publicação:
2024
Tipo de documento:
Article