Optimization of a protein synthesizing lysate system from Trypanosoma cruzi

Abstract

A protein-synthesizing lysate system from Trypanosoma cruzi epimastigotes analogous to the rabbit reticulocyte lysate system was established. The system was optimized by the ‘classical’ method where one of the factors is varied while the others are kept constant. With this the following optima were found: [Mg 2+]: 1.0 mM, [K +]: 60 mM, T: 25°C, pH: 7.5. This method was compared with the ‘sequential simplex’ method [Long, D.E. (1969) Anal. Chim. Acta 46, 93–100], a method designed to optimize rationally interdependent factors in biological systems. The optima as determined with this method were: [Mg 2+]: 1.02 mM, [K +]: 63 mM, T: 25.5°C, pH: 7.25. At these values the system incorporated 43% more amino acids into proteins than a system optimized with the ‘classical’ method. Fluorographic analysis of the proteins synthesized by the system shows that while proteins in the molecular weight range between 14 000 and 45 000 are synthesized in amounts comparable to the in vivo situation, the higher molecular weight proteins (>45 000) are synthesized in lesser quantities.