Hatano, T., Alioto, S., Roscioli, E., Palani, S., Clarke, S. T., Kamnev, A., … Balasubramanian, M. K. (2018). Rapid production of pure recombinant actin isoforms in Pichia pastoris. Journal of Cell Science. doi: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.
License type:
Attribution 4.0 International (CC BY 4.0)
Funding Info:
This work was supported by Wellcome Trust Senior Investigator Award (WT101885MA), a Wellcome Trust Collaborative Award in Science (203276/Z/16/Z), a Royal Society Wolfson merit award (WM130042) and an European Research Council Advanced Grant (ERC-2014-ADG No. 671083) to M.K.B. B.G. was supported by a grant from the National Institutes of Health (GM063691). A.D.M. was supported by a Wellcome Trust Senior Investigator Award (grant 106151/Z/14/Z) and a Royal Society Wolfson Research Merit Award (grant WM150020). K.S. was funded by the Biotechnology and Biological Sciences Research Council (BB/L007525/1) and Wellcome Trust Warwick ISSF Quantitative Biomedicine Programme and Warwick Research Development Fund awards. R.C.R. was supported by core funding from Institute of Molecular and Cell Biology (IMCB)/Agency for Science, Technology and Research (A*STAR). S.T.C. was funded by a studentship from the Engineering and Physical Sciences Research Council. Deposited in PMC for immediate release.