Recombinant microtubule (MT)-severing proteins are valuable for studying their mechanisms of action
however, purifying them in an active state is challenging. Here, we provide a protocol to obtain biologically active and highly pure recombinant GFP-UNC-45A, a novel ATP-independent MT-severing protein, from E. coli. We describe steps for using GST-affinity and spin columns and detail procedures for assessing the activity of GFP-UNC-45A with in vitro MTs along with GFP-katanin as a positive control. The purified proteins can be used for downstream applications to study their functions. For complete details on the use and execution of this protocol, please refer to Habicht et al.