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

Abstract

Actin is one of the most abundant eukaryotic cytoskeletal polymer-forming proteins, which in the filamentous form regulates a number of physiological processes, ranging from cell division and migration to development and tissue function. Actins are differentially posttranslationally modified (PTMs) in different organisms, which include Met, Ala, Asp, and Glu N-acetylation, N-arginylation, and the 73th His residue (His-73) methylation, with different organisms displaying a distinct signature of PTMs. Currently methods are not available to produce actin isoforms with organism specific PTM profile. Here we report Pick-ya actin, a method to express actin isoforms from any eukaryote with its own key characteristic PTM pattern. We achieve this using a synthetic biology strategy in a yeast strain that expresses 1. actin isoforms with the desired N-end via ubiquitin fusion and 2. mammalian enzymes that promote acetylation and methylation. Pick-ya actin should greatly facilitate biochemical, structural, and physiological studies of the actin cytoskeleton and its PTMs. Materials and Methods Plasmids and strainsPlasmids and strains used in this study are listed in Table S1 and S2, respectively. pPICZc-actin-thymosin β4-8HisActin-coding sequences codon-optimized for expression in P. pastoris were synthesized by gBlocks (IDT) and cloned into pPICZc (Invitrogen), which adds, at the C-terminus, an in-frame chymotrypsin cleavage site, linker sequence, thymosin β 4 and a His tag (Noguchi et al., 2007; Hatano et al., 2018). N-terminal ubiquitin fusion constructsFive' part of S. cerevisiae UBI4 gene encoding ubiquitin repeat 1 (Ubi4 1-76 amino acid residues) were PCR amplified from genomic DNA isolated from S.cerevisiae and cloned at the 5' end of in-frame actin in pPICZc-actin-thymosin β4-8His linearized by EcoRI using Gibson assembly. The first ATG for actin was removed by the cloning.NAA80 and SETD3 genes NAA80 and myc-SETD3 genes codon-optimized for expression in P. pastoris were synthesised by gBlocks (IDT) and cloned into pIB2 (PGAP promoter) or pIB4 (PAOX1 promoter) P. pastoris expression vectors by Gibson assembly. Dual expression of NAA80 and myc-SETD3A PCR-amplified PGAP:NAA80::TAOX1 DNA fragment was cloned at the 3' of NAA80 gene. The resulting plasmid lacking terminator for NAA80 gene was linealised by PstI/SphI digestion and CYC1 terminator was cloned by ligation. P. pastoris cultureThe composition of the minimal glycerol (MGY) and minimal methanol (MM) growth media and basic techniques for P. pastoris are described in the Pichia Expression Kit Instruction Manual (https://www.thermofisher.com/order/catalog/product/V19020). P. pastoris transformationPlasmid DNA was linearized and yeast cells were transformed by using the lithium chloride method or electroporation. Transformants with pPICZc plasmid were selected on yeast extract peptone dextrose (YPD) solid media containing 100 mg/l Zeocin (Gibco, #R25001). For transformation of cells with pIB2 or pIB4 plasmids, his4 cells were used as the host and transformants were se...

Purification of Human β- and γ-actin from Budding Yeast

“…Although N-terminal differences may be important for some applications, they appear to be less consequential for others (Cook et al, 1992;Hatano et al, 2018). Thus, should the strict nature of PTMs at or near the N-terminus of actin become "mission critical", an alternative Pichia-based system may prove advantageous or a useful partner to our method (Hatano et al, 2020).…”

“…While these and related purification methods have been adopted for various studies of normal and mutant versions of actin, preparations are often contaminated with low amounts (5-15%) of host actin (Hundt et al, 2014;Muller et al, 2013;Muller et al, 2012;von der Ecken et al, 2016). Other systems use a combination of affinity tags and a direct fusion to the actin monomer binding protein thymosin-β4 (Tβ4) to prevent the spurious polymerization or aggregation of recombinant actin and to facilitate isoform specific purification (Hatano et al, 2018;Hatano et al, 2020;Kijima et al, 2016;Noguchi et al, 2007). With additional modifications this approach permits the isolation of actin isoforms and of specific post translational states: Nacetylation, N-arginylation, and methylation of a conserved histidine residue (Hatano et al, 2018;Hatano et al, 2020).…”

Diversity from similarity: cellular strategies for assigning particular identities to actin filaments and networks

“…However, we still do not have a satisfying overview of the variety of possible differences among all actin isoforms expressed in eukaryotes. Recently, new protocols have been developed [ 92 – 94 ], allowing for a wider variety of actin isoforms to be purified. It is likely that future comparisons of a greater diversity of actin isoforms, purified from similar protocols, will strengthen our knowledge of these mechanisms.…”

Tools of the trade: studying actin in zebrafish

“…The labelled actins can be introduced into zebrafish embryos via microinjection (Cheng et al 2004 ). Recently, a robust new method for the expression of homogenous functional actins from recombinant yeast has been developed (Hatano et al 2018 , 2020 ). Actin expressed, purified and labelled in this way has been tested in zebrafish embryos (Hatano et al 2018 ).…”