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RasIns: Genetically Encoded Intrabodies of Activated Ras Proteins.

Abstract：

:K- and H-Ras are the most commonly mutated genes in human tumors and are critical for conferring and maintaining the oncogenic phenotype in tumors with poor prognoses. Here, we design genetically encoded antibody-like ligands (intrabodies) that recognize active, GTP-bound K- and H-Ras. These ligands, which use the 10th domain of human fibronectin as their scaffold, are stable inside the cells and when fused with a fluorescent protein label, the constitutively active G12V mutant H-Ras. Primary selection of ligands against Ras with mRNA display resulted in an intrabody (termed RasIn1) that binds with a KD of 2.1μM to H-Ras(G12V) (GTP), excellent state selectivity, and remarkable specificity for K- and H-Ras. RasIn1 recognizes residues in the Switch I region of Ras, similar to Raf-RBD, and competes with Raf-RBD for binding. An affinity maturation selection based on RasIn1 resulted in RasIn2, which binds with a KD of 120nM and also retains excellent state selectivity. Both of these intrabodies colocalize with H-Ras, K-Ras, and G12V mutants inside the cells, providing new potential tools to monitor and modulate Ras-mediated signaling. Finally, RasIn1 and Rasin2 both display selectivity for the G12V mutants as compared with wild-type Ras providing a potential route for mutant selective recognition of Ras.

journal_name

J Mol Biol

journal_title

Journal of molecular biology

authors

Cetin M,Evenson WE,Gross GG,Jalali-Yazdi F,Krieger D,Arnold D,Takahashi TT,Roberts RW

doi

10.1016/j.jmb.2016.11.008

subject

Has Abstract

pub_date

2017-02-17 00:00:00

pages

562-573

issue

4

eissn

0022-2836

issn

1089-8638

pii

S0022-2836(16)30490-9

journal_volume

429

pub_type

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