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.
Original language | English (US) |
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Pages (from-to) | 434-440 |
Number of pages | 7 |
Journal | Analytical biochemistry |
Volume | 99 |
Issue number | 2 |
DOIs | |
State | Published - Nov 1 1979 |
ASJC Scopus subject areas
- Biophysics
- Biochemistry
- Molecular Biology
- Cell Biology