OSMO-EXPRESSION AND FAST 2-STEP PURIFICATION OF ESCHERICHIA-COLI TRANSLATION TERMINATION FACTOR RF-3

The gene for the translation termination factor RF-3 in Escherichia co li has recently been cloned and sequenced. Only small amounts of the p rotein have been purified until now, not sufficient for detailed inves tigation of the structure and function of this factor. For such studie s, we have developed an overexpression system and a purification proce dure suitable for large quantities of RF-3. The gene prfC was cloned i nto the osmo-inducible plasmid pOSEX3 and subsequently transformed int o the E. coli strain MKH13. The expression of prfC in this plasmid, wh ich is under the control of the osmotic pressure in the growth medium, Leads to a level of RF-3 more than 100-times higher than that in wild -type cells. Using a new two-step FPLC protein purification procedure consisting of ion-exchange chromatography on Q-Sepharose FF and S-Seph arose HP, we obtain 220 mg pure RF-3 from 10 g overproducing cells, co rresponding to 55 mg RF-3/1 medium. The identity of the purified prote in was confirmed by matrix-assisted laser desorption/ionisation mass s pectrometry of tryptolytic fragments and by N-terminal amino acid sequ encing. The activity of the purified factor was tested in vitro by mea suring the stimulation of RF-2 dependent formylmethionine release from a ribosomal termination complex and the binding capacity of GTP and G DP. All assays showed that the purified RF-3 was highly active with a specific activity of approximately 2000 units/mg.