Production and Membrane Binding of N-Terminally Acetylated, C-Terminally Farnesylated and Carboxymethylated KRAS4b.
Methods Mol Biol
; 2262: 105-116, 2021.
Article
in En
| MEDLINE
| ID: mdl-33977473
ABSTRACT
Recombinant mammalian proteins are routinely produced in E. coli and thus lack post-translational modifications. KRAS4b is processed at both the N- and C-terminus, resulting in an acetylation of the N-terminus (at Thr, after aminopeptidase removal of the original N-term Met) and farnesylation/carboxymethylation of the C-terminal Cys (after proteolytic cleavage of the original C-terminal three amino acids, Val-Iso-Met). Processing of KRAS enables it to associate with the plasma membrane and fulfill its function in cell signaling. We describe here the production of recombinant KRAS4b from our modified baculovirus/insect cell expression system that accurately incorporates these in vivo modifications to allow experiments that anchor KRAS4b to membrane mimetics (e.g., nanodiscs and liposomes).
Key words
Full text:
1
Collection:
01-internacional
Database:
MEDLINE
Main subject:
Recombinant Proteins
/
Cell Membrane
/
Protein Processing, Post-Translational
/
Proto-Oncogene Proteins p21(ras)
/
Protein Prenylation
Limits:
Humans
Language:
En
Journal:
Methods Mol Biol
Year:
2021
Document type:
Article