Rapid production of pure recombinant actin isoforms in Pichia pastoris

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 Saccharomyces cerevisiae and Schizosaccharomyces 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 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.

Pick-ya actin: A method to purify actin isoforms with bespoke key post-translational modifications

“…Human β and γ actin expressed in P.pastoris are not processed at the N -terminus, as a result methionine is retained at the N -terminus and is acetylated by native acetyl transferases (Hatano et al, 2018). The canonical N -acetylation occurs on Asp residue on β actin and Glu residue on γ actin.…”

“…The canonical N -acetylation occurs on Asp residue on β actin and Glu residue on γ actin. To facilitate generation of β and γ actin with Asp or Glu at the N -terminus, we expressed a DNA molecule (Figure 2A) encoding a translational fusion of ubiquitin with the actin gene lacking the initiator methionine followed by sequences encoding human thymosin β4 (Tβ4) and the 8 consecutive His amino acid residues (8His), which facilitates one-step affinity purification using nickel-NTA resin (Hatano et al, 2018; Huang et al, 2016; Kijima et al, 2016; Noguchi et al, 2007). In this strain, we introduced an integrative plasmid vector backbone or a plasmid expressing human NAA80, which promotes actin N -acetylation.…”

“…In this strain, we introduced an integrative plasmid vector backbone or a plasmid expressing human NAA80, which promotes actin N -acetylation. The translational fusion was purified as described previously using an affinity-binding approach (Hatano et al, 2018). When the four purified actin-fusions were subjected to mass spectrometry, we found that the majority of N -terminus in β actin contained an Asp residue, whereas a large number of Ac-Asp peptides were detected when NAA80 was co-expressed in these cells (Figure 2B and D).…”

“…Thus, precise biochemical and biophysical understanding of actin function in various organisms and tissues depends on the ability to produce actin isoforms with organism-specific signature PTMs. The current methods for actin purification either constitutively introduce Asp/Glu N -acetylation and His-73 methylation (purification from rabbit or chicken muscle, Sf9-insect cell cultures, Dictyostelium , and human platelets) or do not introduce either of the above-mentioned modifications ( Saccharomyces cerevisiae and Pichia pastoris ) (Hatano et al, 2018; Müller et al, 2013; Noguchi et al, 2007; Takashi et al, 2009).…”

“…Here we express NAA80 and SETD3 either singly or together in P.pastoris . This combined with our previously described ubiquitin-fusion approach (Hatano et al, 2018) has led to the development of Pick-ya actin, which facilitates production of actin isoforms with organism specific PTM profiles.…”