Optimisation of protein synthesis rates in reticulocyte lysates and reconstituted systems

Means of increasing the very low activity of a reconstituted protein synthesising system from rabbit reticulocytes were investigated. Increasing the concentration of labelled amino acid, addition of polyamines, use of Sepharose-filtered as opposed to centrifuged ribosomes, use of untreated as opposed to gel-filtered cytosol and an increase in ratio of cytosol to ribosomes all contributed to the increase in activity of the system to the point where activity was clearly consistent with initiation taking place. Similar activities could not be attained with rat liver cytosol though rat liver ribosomes incorporated well in reticulocyte cytosol. Incorporation by lysates was also found to be dependent on the concentration of the labelled amino acid added.