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Purification of eukaryotic cytoplasmic elongation factor 2 and organellar elongation factor G by an affinity binding procedure

Authors
Journal
Analytical Biochemistry
0003-2697
Publisher
Elsevier
Publication Date
Volume
99
Issue
2
Identifiers
DOI: 10.1016/s0003-2697(79)80029-9
Disciplines
  • Biology

Abstract

A rapid affinity binding procedure for obtaining highly purified organellar elongation factor G (EF-G) and cytoplasmic elongation factor 2 (EF-2) in excellent yields from whole cell extracts is described. The procedure involves the addition of ribosomes, GTP, and fusidic acid to a crude cell extract. The fusidic acid stabilizes the formation of a translocase—GDP—ribosome complex which can be recovered from the extract by high-speed centrifugation. The translocase is then released from the ribosomes by a high salt wash. To purify organellar EF-G, 70 S ribosomes from Escherichia coli are used. If 80 S ribosomes such as those from wheat germ are used instead of 70 S ribosomes, EF-2 can be selectively purified from the cell extract. This procedure has been applied to the purification of both cytoplasmic and chloroplast translocases of Euglena gracilis. Chloroplast EF-G and cytoplasmic EF-2 can be separated from each other and from the vast majority of the proteins in the postribosomal supernatant with yields in both cases of 60% or greater.

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