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Cytoskeleton Dynamics pp 19–26Cite as

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

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Part of the book series: Methods in Molecular Biology ((MIMB,volume 2101))

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.

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Acknowledgment

This work is supported by 1000 Youth Talents Program to K.J. and an NSFC grant (31871356) to K.J.

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Authors and Affiliations

  1. The State Key Laboratory Breeding Base of Basic Science of Stomatology (Hubei-MOST) and Key Laboratory of Oral Biomedicine (Ministry of Education), School and Hospital of Stomatology, Wuhan University, Wuhan, China

    Shasha Hua & Kai Jiang

  2. Medical Research Institute, Wuhan University, Wuhan, China

    Shasha Hua & Kai Jiang

Authors
  1. Shasha Hua
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  2. Kai Jiang
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Corresponding author

Correspondence to Kai Jiang .

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Editors and Affiliations

  1. i3S University of Porto, Porto, Portugal

    Helder Maiato

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Hua, S., Jiang, K. (2020). Expression and Purification of Microtubule-Associated Proteins from HEK293T Cells for In Vitro Reconstitution. In: Maiato, H. (eds) Cytoskeleton Dynamics. Methods in Molecular Biology, vol 2101. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-0219-5_2

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  • DOI: https://doi.org/10.1007/978-1-0716-0219-5_2

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-0218-8

  • Online ISBN: 978-1-0716-0219-5

  • eBook Packages: Springer Protocols

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