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Rapid production of pure recombinant actin isoforms in Pichia pastoris
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Hatano, Tomoyuki, Alioto, Salvatore, Roscioli, Emanuele, Palani, Saravanan, Clarke, Scott T., Kamnev, Anton, Hernandez Fernaud, Juan Ramon, Sivashanmugam, Lavanya, Chapa y Lazo, Bernardo, Jones, Alexandra M., Robinson, Robert C., Sampath, Karuna, Mishima, Masanori, McAinsh, Andrew D., Goode, Bruce L. and Balasubramanian, Mohan K. (2018) Rapid production of pure recombinant actin isoforms in Pichia pastoris. Journal of Cell Science, 131 (8). jcs213827. doi:10.1242/jcs.213827 ISSN 1477-9137.
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WRAP-rapid-production-pure-recombinant-actin-isoforms-in-Pichia-pastoris-Jones-2018.pdf - Published Version - Requires a PDF viewer. Available under License Creative Commons Attribution. Download (3988Kb) | Preview |
Official URL: http://dx.doi.org/10.1242/jcs.213827
Abstract
Actins are major eukaryotic cytoskeletal proteins, which perform many important cell functions, including cell division, cell polarity, wound healing, and muscle contraction. Despite obvious drawbacks, muscle actin, which is easily purified, is used extensively presently for biochemical studies of actin cytoskeleton from other organisms / cell types. Here we report a rapid and cost-effective method to purify heterologous actins expressed in the yeast Pichia pastoris. Actin is expressed as a fusion with the actin-binding protein thymosin β4 and purified using an affinity tag introduced in the fusion. Following cleavage of thymosin β4 and the affinity tag, highly purified functional full-length actin is liberated. We purify actins from S. cerevisiae, S. pombe, and the β- and γ- isoforms of human actin. We also report a modification of the method that facilitates expression and purification of arginylated actin, a form of actin thought to regulate actin dendritic networks in mammalian cells. The methods we describe can be performed in all laboratories equipped for molecular biology, and should greatly facilitate biochemical and cell biological studies of the actin cytoskeleton.
Item Type: | Journal Article | ||||||||||||||||||||||||||||||||||||
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Subjects: | Q Science > QH Natural history Q Science > QP Physiology |
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Divisions: | Faculty of Science, Engineering and Medicine > Medicine > Warwick Medical School > Biomedical Sciences > Cell & Developmental Biology Faculty of Science, Engineering and Medicine > Medicine > Warwick Medical School > Biomedical Sciences Faculty of Science, Engineering and Medicine > Science > Life Sciences (2010- ) Faculty of Science, Engineering and Medicine > Medicine > Warwick Medical School |
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Library of Congress Subject Headings (LCSH): | Actin, Eukaryotic cells, Cytoskeleton, Pichia pastoris | ||||||||||||||||||||||||||||||||||||
Journal or Publication Title: | Journal of Cell Science | ||||||||||||||||||||||||||||||||||||
Publisher: | The Company of Biologists Ltd. | ||||||||||||||||||||||||||||||||||||
ISSN: | 1477-9137 | ||||||||||||||||||||||||||||||||||||
Official Date: | 13 March 2018 | ||||||||||||||||||||||||||||||||||||
Dates: |
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Volume: | 131 | ||||||||||||||||||||||||||||||||||||
Number: | 8 | ||||||||||||||||||||||||||||||||||||
Article Number: | jcs213827 | ||||||||||||||||||||||||||||||||||||
DOI: | 10.1242/jcs.213827 | ||||||||||||||||||||||||||||||||||||
Status: | Peer Reviewed | ||||||||||||||||||||||||||||||||||||
Publication Status: | Published | ||||||||||||||||||||||||||||||||||||
Access rights to Published version: | Open Access (Creative Commons) | ||||||||||||||||||||||||||||||||||||
Date of first compliant deposit: | 17 May 2018 | ||||||||||||||||||||||||||||||||||||
Date of first compliant Open Access: | 18 May 2018 | ||||||||||||||||||||||||||||||||||||
RIOXX Funder/Project Grant: |
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