The ribosome as affinity matrix': efficient purification scheme for translation factors.

A convenient method to purify each of the non-ribosomal proteins required to translate a native mRNA in vitro is described. In this scheme, the ribosome is used as an 'affinity' matrix to selectively elute the non-ribosomal proteins required for translation that are bound to these particles. Different sets of these proteins can be eluted with solutions of Mg2+ and NH4+ of various concentrations from either 70S, or 30S and 50S particles. A scheme for the purification of each initiation, elongation and release factor and 20 aminoacyl-tRNA synthetases is described. Specific examples of the purification of the initiation (IF-1, IF-2, IF-3) and elongation (EF-Tu and EF-G) factors and for a protein called 'rescue', which affects the association of native ribosomal subunits, are given. A scheme for the purification of EF-P, which stimulates peptide-bond synthesis and one of the W proteins, which permit reconstitution of translation is also described. The procedure markedly simplifies the isolation, in homogeneous form, of all the non-ribosomal proteins required to reconstruct translation.