Dictyostelium discoideum protein expression

In the slime mould, Dictyostelium discoideum, a STAT-like DNA-binding protein, DIF (the differentiation-inducing factor), is expressed. DIF is about 700 amino-acid residues long, and has substantial homology with mammalian STATs. DIF is, like STAT, activated by phosphorylation of tyrosines. It is essential for differentiation of the stalk cells of D. discoideum A The long evolutionary path that the STATs have travelled from Dictyostelium to mammals underlines their conserved and essential role as signal transmitters and transcriptional activators in early development. 

Dictyostelium discoideum cells can be transformed with plasmid DNA by convenient methods. The first attempts at this, using the calcium phosphate method, were reported in the early 1980s [17, 18]. Protocols were subsequently optimized by Firtel and coworkers [19, 20] and modified in many laboratories (e.g. Ref [21]). Up to 2000 transformants can be recovered from 10 D. discoideum cells [20], with electroporation protocols having been reported [22, 23]. It has also been noted that the choice of transformation method can strongly influence the yield of heterolo-gously expressed proteins [24]. Hence, both calcium co-precipitation and electroporation should be compared to generate optimized expression strains for the production of biopharmaceutical proteins in D. discoideum. 

To date the firefly luciferase gene has been expressed successfully in a large number of cell lines from different organisms (Table 1). Since extracts are likely to contain ATP, the best way to measure luciferase in these samples is to Inject luciferln as the last substrate. Typically the crude extract is mixed with an excess of ATP-Mg buffered solution which is placed in a lumlnometer and the light reaction is initiated by the injection of luclferin. In assay of cell extracts, ATPases, phosphatases or proteases have not been a serious problem in many of the systems analyzed. Mammalian extracts containing luciferase are stable for at least a month at 4 0, whereas extracts from Dictyostelium discoideum are stable for only a few hours unless protease inhibitors are used (26). Diluted purified luciferase is unstable in the absence of other proteins and loses activity rapidly even at 4 C. The situ detection of luciferase has been reported for plant (16), animal (15), yeast (27), bacterial (27) and Dictyostelium (26) cells, and expression has been observed in both transient infections with DNA and in stable cell lines in plant, animal and Dictyostelium cells (15,16,26). 

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