Purification and bioassay of hedgehog ligands for the study of cell death and survival

The Hedgehog (Hh) family of secreted ligands-composed of Sonic Hedgehog (Shh), Indian Hedgehog (Ihh), and Desert Hedgehog (Dhh)-possesses many roles during embryonic development, adult homeostasis, and cancer. The specific functions of the Hh proteins are intertwined with their requirement as survival factors in Hh-responsive cells. However, studies designed to dissect the anti-apoptotic role of Hhs have been hindered by the lack of simple approaches to purify large quantities of recombinant ligands in the average laboratory setting because of the natural modifications of these proteins with palmitic acid and cholesterol. In this chapter, we provide a comprehensive protocol for the expression of Shh, Ihh, and Dhh in Escherichia coli as fusion proteins with calmodulin-binding peptide to allow easy and rapid purification. The ligands are engineered with a new N-terminus containing two isoleucine residues to provide an essential hydrophobic interphase for achieving high biologic activity. The protocol includes a detailed description of a method for determination of the specific activity of the generated proteins by use of a cell culture-based luciferase approach.