Expression and Purification of Microtubule-Associated Proteins from HEK293T Cells for In Vitro Reconstitution.

Abstract

In vitro reconstitution has been an invaluable tool to elucidate the roles and mechanisms of microtubule-associated proteins (MAPs). Like all biochemical assays, the quality of the proteins is vital for success. In the microtubule field, proteins produced in bacteria and insect cells have been widely used for in vitro reconstitution. Recently, we applied the mammalian HEK293T cell expression system to our research on several MAPs. We find that such system is especially suitable for quick functional studies and can produce active proteins that sometimes are difficult for either bacteria or insect cell expression systems. Here, we provide a detailed protocol to express and purify microtubule-associated proteins from HEK293T cells using a Strep-tag strategy. The method described here can be adopted for preparation of other proteins and protein complexes for reconstitution studies.