FAST LARGE-SCALE PURIFICATION OF TETRACYCLINE REPRESSOR VARIANTS FROMOVERPRODUCING ESCHERICHIA-COLI STRAINS

We constructed a plasmid for overexpression of Tn10 Tet repressor (Tet R) by placing a synthetic tetR gene under control of the P-tac promote r. Active TetR is expressed up to 30% of the total soluble cell protei n. A protocol containing anion-exchange, cation-exchange, and size-exc lusion chromatography steps is described for the large-scale purificat ion of milligram amounts of TetR in three days. Cation-exchange chroma tography already yields almost homogenous TetR. Purification of about fifty TetR mutants demonstrates that this protocol is generally applic able. No correlation between net charge of TetR variants and elution b ehaviour was detected for the anion-exchange column. On the other hand , TetR mutants with increased negative charge in their DNA binding dom ain eluted at lower NaCl concentration from the cation-exchange column . The applicability of this purification protocol to the wide variety of TetR variants suggests that it can be used for the rapid purificati on of other DNA binding proteins as well.