Fast large-scale purification of tetracycline repressor variants from overproducing Escherichia coli strains.

TitleFast large-scale purification of tetracycline repressor variants from overproducing Escherichia coli strains.
Publication TypeJournal Article
Year of Publication1996
AuthorsEttner N, Müller G, Berens C, Backes H, Schnappinger D, Schreppel T, Pfleiderer K, Hillen W
JournalJ Chromatogr A
Date Published1996 Aug 23
KeywordsAlleles, Ammonium Sulfate, Bacterial Proteins, Base Sequence, Cell Extracts, Chemical Precipitation, Chromatography, Gel, Chromatography, Ion Exchange, DNA Primers, Electrophoresis, Polyacrylamide Gel, Escherichia coli, Gene Expression Regulation, Bacterial, Isopropyl Thiogalactoside, Plasmids, Polymerase Chain Reaction, Reagent Kits, Diagnostic, Recombinant Proteins, Repressor Proteins, Silver Staining, Spectrophotometry, Ultraviolet, Tetracycline

We constructed a plasmid for overexpression of Tn10 Tet repressor (TetR) by placing a synthetic tetR gene under control of the Pc promoter. Active TetR is expressed up to 30% of the total soluble cell protein. A protocol containing anion-exchange, cation-exchange, and size-exclusion chromatography steps is described for the large-scale purification of milligram amounts of TetR in three days. Cation-exchange chromatography already yields almost homogenous TetR. Purification of about fifty TetR mutants demonstrates that this protocol is generally applicable. No correlation between net charge of TetR variants and elution behaviour was detected for the anion-exchange column. On the other hand, TetR mutants with increased negative charge in their DNA binding domain 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 purification of other DNA binding proteins as well.

Alternate JournalJ Chromatogr A
PubMed ID8817886

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