KRAS(G12V) His Tag Protein, Human

KRAS (Kirsten rat sarcoma viral oncogene homolog) is a small GTPase that functions as a molecular switch regulating cell proliferation, differentiation, and survival signaling. Its protein structure consists of an N-terminal catalytic G domain (containing the P-loop, switch I/II regions, and the G12V mutation site) and a C-terminal hypervariable region (CAAX box). The G12V mutation locks the switch II region in a constitutively active GTP-bound state, abolishing GTPase activity and leading to persistent activation of downstream pathways such as RAF-MEK-ERK, PI3K-AKT, and RAL-GEF, thereby driving tumorigenesis. Clinically, KRAS G12V is one of the most common KRAS mutation subtypes in pancreatic cancer (approximately 30%), colorectal cancer, and lung cancer. Due to the lack of a cysteine residue for covalent targeting, it cannot be treated with G12C inhibitors, and therapeutic options have long been limited. In recent years, emerging strategies—including Pan-KRAS inhibitors, PROTAC degraders, mRNA vaccines targeting G12V neoantigens, and TCR-T cell therapies—have achieved breakthroughs, offering new precision treatment directions to overcome this "undruggable" target.

Experimental Methods

Experimental Principles: The GTPase Glo™ assay evaluates KRAS activity by measuring the amount of GTP remaining after hydrolysis in the KRAS reaction.

Experimental Materials

1.KRAS(G12V) His Tag Protein, Human

2.GTPase Glo™ Assay (Promega, Catalog # V7681T)

3.Solid white multi-well plate (384-well plate) (Corning, Catalog #3572)

4.Plate Reader (PerkinElmer)

Experimental Steps

1.Prepare a 2X GTP solution containing 10 µM GTP and 2 mM DTT using GTPase/GAP Buffer.

2.Dilute KRAS with GTPase/GAP Buffer to concentrations of 100 µg/mL, 80 µg/mL, and 60 µg/mL, then add 5 µL to each well of a 384-well plate, respectively.

3.Add 5 µL of the 2X GTP solution prepared in Step 1 to each well to initiate the reaction. Use 5 µL of GTPase/GAP Buffer mixed with the 2X GTP solution as a blank control. The total reaction volume is 10 µL.

4.Incubate the reaction at room temperature (22–25°C) for 30 minutes.

5.Gently invert and mix the thawed GTPase Glo™ Reagent, 500X; do not vortex. Depending on your needs, adjust the volumes of the following components proportionally to prepare the desired volume of reconstituted GTPase Glo™.

6.Add 10 µL of resuspended GTPase-Glo™ Reagent to the reaction system that has completed the reaction, mix briefly, and then incubate with shaking at room temperature (22–25°C) for 30 minutes.

7.Add 20 µL of the detection reagent, and incubate the microplate at room temperature (22–25°C) for 5–10 minutes.

8.Read the results in endpoint mode at an emission wavelength of 555 nm (luminescence).

9.Calculate the specific activity.

Sample OD value: OD value of remaining ATP

Blank OD value: OD value after adding GTP

50 pmol: Amount of GTP added

Incubation time: 30 minutes

Enzyme amounts: 0.5 μg, 0.4 μg, and 0.3 μg