Protocol for purifying biologically active microtubule-severing protein UNC-45A from E.coli using GST-affinity and spin columns
Year: 2025
Type: article
Abstract: 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. 1 • Instructions for purification of microtubule-severing protein UNC-45A with GFP fusion • Guidance on the preparation of microtubules and TIRF chamber • Steps to evaluate the activity of recombinant protein GFP-UNC-45A using TIRF microscopy Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics. 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.
Source: STAR Protocols
Language: English
Subfield: Cell Biology
Domain: Life Sciences
Open Access status: gold
Estimated APC paid: $1,550
OpenAlex ID: W4407979501