Fully Processed Recombinant KRAS4b: Isolating and Characterizing the Farnesylated and Methylated Protein.
J Vis Exp
; (155)2020 01 16.
Article
en En
| MEDLINE
| ID: mdl-32009649
ABSTRACT
Protein prenylation is a key modification that is responsible for targeting proteins to intracellular membranes. KRAS4b, which is mutated in 22% of human cancers, is processed by farnesylation and carboxymethylation due to the presence of a 'CAAX' box motif at the C-terminus. An engineered baculovirus system was used to express farnesylated and carboxymethylated KRAS4b in insect cells and has been described previously. Here, we describe the detailed, practical purification and biochemical characterization of the protein. Specifically, affinity and ion exchange chromatography were used to purify the protein to homogeneity. Intact and native mass spectrometry was used to validate the correct modification of KRAS4b and to verify nucleotide binding. Finally, membrane association of farnesylated and carboxymethylated KRAS4b to liposomes was measured using surface plasmon resonance spectroscopy.
Texto completo:
1
Colección:
01-internacional
Base de datos:
MEDLINE
Asunto principal:
Proteínas Recombinantes
/
Proteínas Proto-Oncogénicas p21(ras)
/
Prenilación de Proteína
Límite:
Animals
Idioma:
En
Revista:
J Vis Exp
Año:
2020
Tipo del documento:
Article