Introduction of additional charges as an aid in protein purification: isolation of elongation factor 2 from Sulfolobus acidocaldarius by preparative isoelectric focusing before and after ADP-ribosylation.
Diphtheria toxin catalyzes the ADP-ribosylation of elongation factor 2 (EF-2) in eukaryotes and archae-bacteria. As the reaction is strictly EF-2 specific and introduces two negative charges into the molecule, the resulting shift in the isoelectric point (pI) by 0.2 pH units was used to establish a new purification method for EF-2 from Sulfolobus acidocaldarius. The cells were lysed with dithiothreitol at pH 9 and EF-2 was purified by ammonium sulfate precipitation, gel filtration on Sephadex G-200, and three isoelectric focusing steps. The EF-2-containing fractions from the first isoelectric focusing step at pH 4-9 were refocused in a more narrow pH-gradient (pH 5-7). The EF-2 peak from the second step was eluted, collecting only the fractions above the pH region where ADP-ribosylated EF-2 would focus. The EF-2 was then ADP-ribosylated with diphtheria toxin and NAD and subjected to further isoelectric focusing (pH 5-7). The EF-2 was almost homogeneous since ADP-ribosylation had shifted it into a region of the pH gradient free of contaminating proteins. Diphtheria toxin was immobilized on CNBr-activated Sepharose to prevent a possible contamination by proteins from the diphtheria toxin preparation which might have the same pI as ADP-ribosylated EF-2. Finally, the ADP-ribosyl group was removed by equilibrium dialysis using diphtheria toxin and nicotinamide at pH 6.3. The obtained EF-2 was active in protein synthesis.
Siegmund
K D
KD
Biochemisches Institut, Christian-Albrechts Universität, Kiel, Germany.
Klink
F
F
D000246:Adenosine Diphosphate Ribose / Q000737:chemistry*; D002850:Chromatography, Gel; D004167:Diphtheria Toxin; D004797:Enzyme-Linked Immunosorbent Assay; D007525:Isoelectric Focusing; D020652:Peptide Elongation Factor 2; D010445:Peptide Elongation Factors / Q000737:chemistry / Q000302:isolation & purification*; D016945:Sulfolobus acidocaldarius / Q000737:chemistry*
D004167:Diphtheria Toxin; D020652:Peptide Elongation Factor 2; D010445:Peptide Elongation Factors; D000246:Adenosine Diphosphate Ribose