Preparation of K-Ras4B containing synthetic modifications via expressed protein ligation

Prenylation is a lipid post-translational modification (PTM) on proteins that plays a critical role in regulating the interaction of proteins with the membrane. Approximately 2 % of proteins in cells are prenylated. Dysregulation of prenylation has been implicated in several diseases where it affects protein localization and function of these proteins in the cell. Ras proteins are members of a broad family of small GTPases that are prenylated and therefore able to localize to the cell membrane and turn on downstream signaling. Because of these roles, inhibiting the prenylation of oncogenic Ras proteins may serve as a method to control Ras mutation-related cancers. To understand the mechanism of Ras protein prenylation and subsequent interactions, chemically modified forms of Ras can be particularly useful. In this chapter, a platform to prepare modified Ras proteins using expressed protein ligation is described. The workflow involves the expression and purification of a truncated Ras thioester protein, the synthesis of a hypervariable region peptide, and the subsequent ligation of these two fragments to obtain the full-length protein.