International Journal of Biological Macromolecules 39 (2006) 37–44 Use of the myosin motor domain as large-affinity tag for the expression and purification of proteins in Dictyostelium discoideum  Martin Kollmar ∗ Abteilung NMR basierte Strukturbiologie, Max-Planck-Institut f¨ ur Biophysikalische Chemie, Am Faßberg 11, D-37077 G¨ ottingen, Germany Received 30 November 2005; received in revised form 17 January 2006; accepted 18 January 2006 Available online 6 March 2006 Abstract The cellular slime mold Dictyostelium discoideum is increasingly be used for the overexpression of proteins. Dictyostelium is amenable to classical and molecular genetic approaches and can easily be grown in large quantities. It contains a variety of chaperones and folding enzymes, and is able to perform all kinds of post-translational protein modifications. Here, new expression vectors are presented that have been designed for the production of proteins in large quantities for biochemical and structural studies. The expression cassettes of the most successful vectors are based on a tandem affinity purification tag consisting of an octahistidine tag followed by the myosin motor domain tag. The myosin motor domain not only strongly enhances the production of fused proteins but is also used for a fast affinity purification step through its ATP-dependent binding to actin. The applicability of the new system has been demonstrated for the expression and purification of subunits of the dynein–dynactin motor protein complex from different species. © 2006 Elsevier B.V. All rights reserved. Keywords: Myosin motor domain; Dictyostelium discoideum; Large-affinity tag 1. Introduction The social amoeba, Dictyostelium discoideum, is an attrac- tive eukaryotic host for recombinant protein expression. In contrast to tissue cultures of mammalian or insect cells, Dic- tyostelium cells grow fast, and large quantities of cells can be produced easily and cheaply in shake cultures or fermentors [1,2]. Dictyostelium is non-pathogenic, requires no serum or other animal proteins for growth, and is capable of performing post-translational protein modifications [3] and secreting high levels of protein [4]. Dictyostelium grows as single cells, but Abbreviations: ARP1, actin-related protein 1; ARP11, actin-related pro- tein 11; N-CBZPA, N-carbobenzyloxy-phenyl-alanine; Dd, Dictyostelium dis- coideum; DynHC, dynein heavy chain; DynIC, dynein intermediate chain; DynLIC, dynein light–intermediate chain; GST, Glutathione S-transferase; Hs, Homo sapiens; MBP, maltose binding protein; Mm, Mus musculus; PBS, phosphate-buffered saline; PMSF, phenylmethyl sulfonyl fluoride; Sc, Saccha- romyces cerevisiae; TAME, tosylarginine methyl ester; TEV, tobacco etch virus; TLCK, 1-chloro-3-tosylamido-7-amino-2-heptanone HCl; TPCK, 1-chloro-3- tosylamido-4-phenyl-2-butanone  Note: This paper was presented at the Challenging Proteins Workshop in Paris, 17–18 October 2005. ∗ Tel.: +49 551 2012235; fax: +49 551 2012202. E-mail address: mako@nmr.mpibpc.mpg.de. a developmental phase can be triggered by starvation, leading to the formation of a multicellular organism, which then dif- ferentiates to produce a fruiting body composed of spores on a stalk. Many transformation vectors exist for the temporal- or spatial-specific expression of mutated or tagged proteins. Three classes of vectors are used for the expression of proteins in Dictyostelium: (A) integrating plasmids (e.g. [5]); (B) plas- mids allowing autonomous Ddp1-based replication (e.g. [6]); (C) plasmids allowing Ddp2-based extrachromosomal replica- tion (e.g. [7]). The latter vectors require the presence of the Ddp2 ORF product for autonomous replication, which is known to be essential in trans for plasmid maintenance, while an approxi- mately 600 bp fragment, presumably containing the Ddp2 origin of replication, is required in cis [8,9]. This can be achieved either by the use of the AX3-ORF + cells that carry integrated copies of the Ddp2 ORF gene, or by co-transformation with plasmids such as pREP, which carry a copy of the ORF gene. To enhance protein production, and to facilitate protein purifi- cation, a similar set of fusion-tags and –proteins that are used in bacteria has been established for Dictyostelium. Initially these vectors contained only multiple cloning sites with few restric- tion sites, and either N- or C-terminal poly-histidine or c-myc tags [7]. This set of vectors was extended by adding Flag-, Strep-, GST- (all reported in [10]), myosin motor domain-tags 0141-8130/$ – see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.ijbiomac.2006.01.005