Expression of protein tyrosine kinase pp60v-src variants in Dictyostelium discoideum.
We achieved production of v-Src of the low-oncogenic PRC and its variant proviral structure H19 in Dictyostelium discoideum, an emerging host system suitable for synthesis of heterologous proteins. To accomplish their expression, the first six codons of the N-terminus of v-src had to be changed according to the D. discoideum codon preference. Alternatively, N-terminal fusions of 6xHis-tag or GFP were sufficient to overcome the incompatibility in codon usage. D. discoideum-expressed v-Src kinases of the expected molecular weight were recognized by Src-specific antibodies; GFP-PRC was distributed uniformly in the cytosol. In contrast to other lower eukaryotes, where the accumulation of v-Src leads to growth inhibition, D. discoideum cells silenced the kinase activity of PRC-derived v-Src and showed no developmental or growth defects.
Brábek
J
J
Department of Physiology, Charles University, Prague, Czech Republic.
Mojzita
D
D
Půta
F
F
D000818:Animals; D001358:Avian Sarcoma Viruses / Q000235:genetics; D002846:Chromatography, Affinity; D004023:Dictyostelium / Q000235:genetics*; D015870:Gene Expression; D016391:Genes, src / Q000235:genetics*; D005822:Genetic Vectors; D008856:Microscopy, Fluorescence; D015688:Oncogene Protein pp60(v-src) / Q000096:biosynthesis* / Q000235:genetics* / Q000302:isolation & purification
D015688:Oncogene Protein pp60(v-src)