Separation of nucleic acids and proteins

Abstract

This chapter focuses on a unique problem encountered during recovery of intracellulary produced proteins. Disruption of cells produces a mixture of nucleic acids and proteins in the solution from which the desired proteins must be fractionated. Typical separation schemes involve first the removal of nucleic acids from solution by precipitation. The desired protein is then isolated and purified from the mixture of remaining nucleic acids and proteins. A scheme for recovery of intracellular bacterial enzyme tartrate dehydrogenase from cell paste is shown in the chapter. Material balance at the different stages of the scheme in two different experiments showed that 53-60% loss in enzyme activity took place during precipitation of nucleic acids by protamine sulfate and during ammonium sulfate fractionation of proteins. Reduction in volume, removal of major nonprotein impurities, and little increase in fold purification of the desired enzyme characterize these steps. Accordingly, these steps represent the classical isolation steps. Thus, the chapter discusses the aqueous two-phase extraction that is an efficient method for separation of nucleic acids from proteins. By proper selection of an ATP system and optimization of pH and salt concentration, it is possible to achieve a high degree of purification using a single-stage extraction-back extraction for an intracellular enzyme. The enzyme yield and purification achieved by ATP process is significantly higher than from a comparative precipitation process.