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. 2018 Apr 23;131(8):jcs213827.
doi: 10.1242/jcs.213827.

Rapid production of pure recombinant actin isoforms in Pichia pastoris

Tomoyuki Hatano  1 , Salvatore Alioto  2 , Emanuele Roscioli  3 , Saravanan Palani  3 , Scott T Clarke  3 , Anton Kamnev  3 , Juan Ramon Hernandez-Fernaud  4 , Lavanya Sivashanmugam  3 , Bernardo Chapa-Y-Lazo  3 , Alexandra M E Jones  4 , Robert C Robinson  5   6   7 , Karuna Sampath  3 , Masanori Mishima  3 , Andrew D McAinsh  3 , Bruce L Goode  2 , Mohan K Balasubramanian  1
Affiliations

Rapid production of pure recombinant actin isoforms in Pichia pastoris

Tomoyuki Hatano et al. J Cell Sci. .

Abstract

Actins are major eukaryotic cytoskeletal proteins, and they are involved in 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 for biochemical studies of the non-muscle actin cytoskeleton. 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 by means of 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 Saccharomycescerevisiae and Schizosaccharomycespombe, 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 dendritic actin 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.

Keywords: Actin; Actin purification; Biochemistry; Cytoskeleton.

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Conflict of interest statement

Competing interestsThe authors declare no competing or financial interests.

Figures

Fig. 1.
Fig. 1.
Purification of assembly-competent recombinant Saccharomyces cerevisiae Act1 from Pichia pastoris. (A) Diagram of the actin purification method (for details see Materials and Methods). (B) Coomassie-stained gel (CBB) showing purified recombinant Saccharomyces cerevisiae Act1. Numbering (1–6) at the bottom corresponds to the steps designated by numbers in the flow chart in A. (C) TIRF microscopy analysis of actin filament growth, comparing Act1 purified from S. cerevisiae (native) and recombinant Act1 (recombinant) purified from P. pastoris, in the presence or absence of S. cerevisiae profilin (Pfy1) and formin (Bnr1). All reactions contained 10% Oregon Green-labelled skeletal muscle actin as a tracer. Data are averaged from three independent experiments (n=10 filaments per experiment; 30 total). Error bars show the s.e.m. (D) Actin filament elongation rates calculated from reactions in C. n.s., not significant (Student's t-test).
Fig. 2.
Fig. 2.
Purification of recombinant human non-muscle actins and fission yeast Act1. (A) CBB gel image showing purified recombinant human β-actin (Actb), human γ-actin (Actg1) and Schizosaccharomyces pombe (SpAct1) actin. Immunoblotting in the bottom panels was performed with antibodies against Actb or Actg1 raised against peptides corresponding to the N-terminus of human β- and γ-actins. Since the N-terminus of S. pombe Act1 has strong similarity to human γ-actin, the anti-Actg1 antibody can recognize S. pombe Act1 as well as γ-actin (see sequence alignment shown at the bottom). (B) Actin sedimentation assays. Purified actin in low-ionic strength buffer (G-buffer) was mixed with KCl, EGTA and Mg2+ to induce actin polymerization. Actin sedimentation was tested in the presence of 0.5 mM LatA or in solvent control (DMSO) and in the absence of DMSO or Latrunculin A (‘−’). After ultracentrifugation, actin in the pellet (Pellet) and supernatant (Sup) fractions were detected by CBB staining following SDS-PAGE. Rabbit muscle actin was used as a positive control. (C) Visualization of F-actin. The specified actins were incubated with KCl, EGTA and Mg2+ in the presence of 2% PEG. F-actin in all cases was then stained with Rhodamine–phalloidin and imaged by using a Nikon inverted microscope fitted with a Yokogawa spinning-disc head and an Andor iXon camera. Scale bars: 5 µm.
Fig. 3.
Fig. 3.
Recombinant actins were incorporated into actin cytoskeleton networks in cells. (A,B) Schematic representation and gel image of labelling method for actin Cys374 (details are shown in Materials and Methods). Eluates collected after chymotrypsin treatment (see Fig. 1A) were mixed with KCl and Mg2+ to induce polymerization of β-actin (lane 1) and incubated with Alexa Fluor 488 C5 maleimide to label the residue 374 of β-actin in F-actin (lane 2). The reaction mixture was spun in an ultracentrifuge to pellet F-actin [lane 3, supernatant (Sup); lane 4, pellet fraction (Pellet)]. The pellet fraction was suspended in low-salt G-buffer, and dialyzed against the same G-buffer. The dialyzed sample (lane 5) was ultracentrifuged to remove F-actin (lane 6). Free Alexa Fluor 488 C5 maleimide was removed by means of a desalting column (lane 7, G-25). (C) Microinjection of β-actin (i) and γ-actin (ii) labelled with TMR into zebrafish embryos. Images were acquired using a spinning-disc confocal microscope. Scale bars: 100 µm. (D) Injection of fluorescently labelled β- and γ-actins into RPE1 cells. β- and γ-actin were labelled with Alexa Fluor 488 and TMR, respectively and injected into human RPE1 cells. The left panel shows a maximum projection of all z-axis stacks. The right panel shows a maximum projection of z-axis stacks 24–26.
Fig. 4.
Fig. 4.
Purification of arginylated β-actin. (A) Schematic representation of expression of arginylated β-actin (R-Actb). (B) Immunoblotting and CBB staining showing purified R-Actb. Immunoblotting was performed with polyclonal antibodies against arginylated β-actin. (C) Representative mass spectra of the unique N-terminal tryptic peptide of β-actin, γ-actin and arginylated β-actin [(R) β-actin] as obtained after HCD fragmentation. The N-terminal (R) β-actin tryptic unique peptide shows the replacement of Met-Asp by Arg. (D) Visualization of arginylated actin (R-Actb) filament. R-Actb was incubated with KCl, EGTA and Mg2+ in the presence of 2% PEG and stained with Cy5–phalloidin. Scale bar: 5 µm.
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