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...