Efficient separation of Thermus aquaticus EF-Tu functional complexes.
A new method for fast separation of the main functional complexes of the elongation factor Tu from Thermus aquaticus has been developed. Binary complexes EF-Tu * GDP and EF-Tu * GDPNP as well as the ternary complex EF-Tu * GDPNP * Leu approximately tRNA were separated from each other by means of HPLC on a hydrophobic sorbent TSK-Gel Phenyl 5PW in a reverse gradient of ammonium sulfate. This technique is suitable for monitoring EF-Tu activity, characterisation of the ratio between different EF-Tu forms in cell extracts, and isolation of individual EF-Tu complexes for structural and functional investigations. In order to illustrate the potentials of the method, we used HPLC on a TSK-Gel Phenyl 5PW matrix to determine the ratio between affinities of GDP and GDPNP for EF-Tu. We found that K(a)(GDP) is about 27 times higher than K(a)(GDPNP) at 37 degrees C, the value being close to the one reported for Thermus thermophilus EF-Tu.
Institute of Molecular and Structural Biology, Aarhus University, Gustav Wieds Vej 10C, Aarhus C, DK-8000, Denmark.
V G
VG
Stepanov
J
J
Nyborg
D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D010444:Peptide Elongation Factor Tu / Q000302:isolation & purification*; D013056:Spectrophotometry, Ultraviolet; D013824:Thermus / Q000737:chemistry*
D010444:Peptide Elongation Factor Tu