Dictyostelium discoideum as protein expression system

The social amoeba, Dictyostelium discoideum, is an attractive eukaryotic host for recombinant protein expression. In contrast to tissue cultures of mammalian or insect cells, Dictyostelium 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 a developmental phase can be triggered by starvation, leading to the formation of a multicellular organism, which then differentiates 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.


Characteristics Escherichia coli Yeast D.discoideum Insect cells
Cell growth Hours to days Days to 1 week Days to 1 week Days to 1 week
Cost of growth medium Low to medium Low to medium Low to medium High
Expression level Low to high Low to high Low to high Low to high
Protein folding Refolding usually be required Refolding might be required Proper folding Proper folding
N-linked glycosylation None High mannose, no sialic acid, non-human sugars added Mammalian-type core Complex, no sialic acid, non-human sugars added
O-linked glycosylation, phosphorylation, acetylation, acylation No Yes Yes Yes

[1]S-I Han, K Friehs, E Flaschel: Improvement of a synthetic medium for Dictyostelium discoideum. Process Biochem. 2004, 39:925-30.
[2]M. Sussman: Cultivation and synchronous morphogenesis of Dictyostelium under controlled experimental conditions. Methods Cell Biol. 1987, 28:9-29.
[3]JC Heikoop, PD Grootenhuis, M Blaauw, JS Veldema, PJ Van Haastert, MH Linskens: Expression of a bioactive, single-chain choriogonadotropin in Dictyostelium discoideum. Eur. J. Biochem. 1998, 256:259-63.
[4]W. Dittrich, K.L. Williams, M.B. Slade: Production and secretion of recombinant proteins in Dictyostelium discoideum. Biotechnology (N Y) 1994, 12:614-18.

The Myosin Fusion System

To enhance protein production, and to facilitate protein purification, 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 restriction sites, and either N- or C-terminal poly-histidine or c-myc tags [1]. This set of vectors was extended by adding Flag-, Strep-, GST- [all reported in 2], myosin motor domain- [3], and MBP-tags [4] as expression and purification aids. A tag only used in Dictyostelium is the motor domain of class II myosin as an N-terminal fusion construct. Class II myosin is one of the most abundantly expressed proteins in Dictyostelium. It has been shown that the motor domain not only enhances the expression of C-terminally fused Dictyostelium proteins like a-actinin [3] or the GTPase domain of dynaminA [5], but also increases expression levels of heterologous protein such as the GTPase domain of rat dynamin1 [6]. In addition to its expression-enhancing capabilities, the myosin motor domain provides a very fast and specific affinity purification step through its ATP-dependent binding to actin (Figure 1). Furthermore, the development of appropriate mild lysis conditions has strongly reduced proteolysis problems that are commonly encountered during the extraction procedure for many cells. We have designed new vectors especially for the purpose to express proteins suitable for structural studies (Figure 2)[7]. 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 vectors have been designed that the expressed proteins do not contain any extensions remaining from cloning sites and that both tags can optionally be cleaved. 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 [7].

Myosin fusion system pDXA-mako2b
Figure 1

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Figure 2

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[1]DJ Manstein, HP Schuster, P Morandini, DM Hunt: Cloning vectors for the production of proteins in Dictyostelium discoideum. Gene 1995, 162:129-34.
[2]ML Knetsch, G Tsiavaliaris, S Zimmermann, U Ruhl, DJ Manstein: Expression vectors for studying cytoskeletal proteins in Dictyostelium discoideum. J. Muscle. Res. Cell. Motil. 2002, 22:605-11.
[3]W Kliche, S Fujita-Becker, M Kollmar, DJ Manstein, FJ Kull: Structure of a genetically engineered molecular motor. Embo J. 2001, 20:40-6.
[4]R. Graf: Maltose-binding protein as a fusion tag for the localization and purification of cloned proteins in Dictyostelium. Anal Biochem. 2001, 289:297-300.
[5]HH Niemann, ML Knetsch, A Scherer, DJ Manstein, FJ Kull: Crystal structure of a dynamin GTPase domain in both nucleotide-free and GDP-bound forms. Embo J. 2001, 20:5813-21.
[6]TF Reubold, S Eschenburg, A Becker, FJ Kull, DJ Manstein: A structural model for actin-induced nucleotide release in myosin. Nat. Struct. Biol. 2003, 10:826-30.
[7]M Kollmar: Use of the myosin motor domain as large-affinity tag for the expression and purification of proteins in Dictyostelium discoideum. Int J Biol Macromol. 2006, 39:37-44.
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