Optimal conditions for cell-free protein synthesis using post-mitochondrial supernatant preparations from newly fertilized eggs of Drosophila melanogaster have been determined. The most active extracts were prepared using 75 mM KCl in potassium N-2-hydroxyethylpiperazine-N′-2-ethane sulfonate (pH 7.4). The optimal pH of the incubation medium was found to be 7.0. Dithiothreitol was more effective than other thiol reagents in enhancing amino acid incorporation by the system and was used in both the extraction and incubation media. A Mg2+ concentration of 4 mM in the incubation system was found to represent a well-defined optimum. The monovalent cation requirement for optimal incorporating activity of the system is complex: NH4 +, K+ and Na+ were all found to be required at a total concentration of 91 mM. Under the optimal conditions defined, the amino acid-incorporating activity of the system is comparable to the activities of mammalian and other insect systems. The Drosophila system is of interest since it apparently is active in cell-free initiation of polypeptide chains - as indicated by its sensitivity to low concentrations of NaF or aurintricarboxylic acid.
ASJC Scopus subject areas