PMID:9089380
| Citation |
Chinali, G (1997) Isolation of tRNA isoacceptors by affinity chromatography with immobilized elongation factor Tu from Escherichia coli. J. Biochem. Biophys. Methods 34:1-10 |
|---|---|
| Abstract |
Elongation factor Tu (EF-Tu) from E. coli was coupled to activated CH Sepharose 4B. The immobilized EF-Tu maintained most of the guanosine nucleotide binding activity, but its ability to bind aminoacyl-tRNA depended on the type of complex used in the coupling reaction. The EF-Tu.GTP.aminoacyl-tRNA.kirromycin complex yielded an immobilized factor that was much more active in binding aminoacyl-tRNA than that obtained by coupling EF-Tu.GDP or EF-Tu.GDP.kirromycin to CH Sepharose 4B. Like the free factor, the immobilized EF-Tu.GTP did bind aminoacyl-tRNAs, but not unacylated tRNAs or N-acylated-aminoacyl-tRNAs. The antibiotic kirromycin was used to obtain the rapid conversion of EF-Tu.GDP to EF-Tu.GTP and the release of aminoacyl-tRNA from the matrix-bound EF-Tu by GDP. When total tRNA was aminoacylated by one amino acid only, a column of immobilized EF-Tu-GTP.kirromycin allowed the isolation of the aminoacylated tRNA from bulk tRNA. A rapid and nearly quantitative recovery of highly purified tRNA isoacceptors for various amino acids was obtained in one chromatographic step. |
| Links | |
| Keywords |
Chromatography, Affinity/methods; Escherichia coli; Evaluation Studies as Topic; Guanosine Triphosphate; Ligands; Peptide Elongation Factor Tu; Pyridones; RNA, Transfer, Amino Acyl/isolation & purification; Sepharose |
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