Expression and Purification of a Mammalian Protein: Cytosolic Domain of IRE1α from Insect Sf21 Cells

Abstract

AbstractEukaryotic proteins can be expressed in different heterologous systems. However, mammalian proteins in general have specific post-translational processing requirements that may not be fulfilled by a regular bacterial expression system. In this study, we use an insect cell system to express a mammalian protein of interest. Spodoptera frugiperda (Sf21) cells were used in conjunction with a baculoviral expression system to produce the cytosolic domain (CD) of IRE1, an endoplasmic reticulum (ER) stress sensor protein. Inositol Requiring Enzyme 1 (IRE1) is a dual function kinase and endoribonuclease protein that cleaves X-box binding protein (XBP1) mRNA. We used the pFastBac plasmid to insert the coding sequence into a recombinant bacmid shuttle vector which was then used to infect Sf21 cells. The expressed protein was then purified with an MBPTrap column to obtain >85% pure protein.