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.
Ganoza
M C
MC
Banting and Best Department of Medical Research, University of Toronto, Ontario, Canada.
Aoki
H
H
Burkhardt
N
N
Murphy
B J
BJ
D000604:Amino Acyl-tRNA Synthetases / Q000302:isolation & purification; D002845:Chromatography; D015977:Eukaryotic Initiation Factor-1 / Q000302:isolation & purification; D010444:Peptide Elongation Factor Tu / Q000302:isolation & purification; D010445:Peptide Elongation Factors / Q000302:isolation & purification*; D010448:Peptide Initiation Factors / Q000302:isolation & purification*; D039881:Prokaryotic Initiation Factor-2; D039665:Prokaryotic Initiation Factor-3; D014176:Protein Biosynthesis; D012270:Ribosomes / Q000737:chemistry*
D015977:Eukaryotic Initiation Factor-1; D010445:Peptide Elongation Factors; D010448:Peptide Initiation Factors; D039881:Prokaryotic Initiation Factor-2; D039665:Prokaryotic Initiation Factor-3; C020746:factor EF-P; D010444:Peptide Elongation Factor Tu; D000604:Amino Acyl-tRNA Synthetases