KRAS is a membrane-bound GTPase that is thought to be the switch for GDP-GTP regulation. the RAS family (KRAS, NRAS, HRAS) is one of the most common causes of cancer in humans, and mutations occur in KRAS account for 86% of all RAS gene mutations. 2021 Amgen (AMGEN) received accelerated FDA approval for KRAS G12C inhibitor Lumakras (sotorasib) to market.

There are numerous mutations in KRAS, besides G12C, common ones include G12D, G12V, G12A and G12S. KRAS G12D has a high mutation rate in pancreatic and colorectal cancers. Many domestic and foreign pharmaceutical companies are also laying out the development of KRAS G12D inhibitors.

KRAS binds to GTP in an activated state and to GDP in an "off" state, which regulates the cytoplasmic signaling pathway and cellular activities.

The conversion of GTP to GDP is an extremely fast process, and the activation state formed by Kras binding to GTP is difficult to obtain.

We expressed and purified the KRAS G12D protein in the "off" state (Fig. 1).

The enzymatic degradation of GDP bound to KRAS was performed by adding GppCp and incubating it with KRAS to obtain KRAS G12D protein in the activated state (Fig. 2).