Fast large-scale purification of tetracycline repressor variants from overproducing Escherichia coli strains.
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.
Ettner
N
N
Lehrstuhl für Mikrobiologie, Institut für Mikrobiologie und Biochemie, Friedrich Alexander Universität Erlangen-Nürnberg, Germany.
Müller
G
G
Berens
C
C
Backes
H
H
Schnappinger
D
D
Schreppel
T
T
Pfleiderer
K
K
Hillen
W
W
D000483:Alleles; D000645:Ammonium Sulfate / Q000737:chemistry; D001426:Bacterial Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D001483:Base Sequence; D002457:Cell Extracts / Q000737:chemistry; D011232:Chemical Precipitation; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D017931:DNA Primers / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D015964:Gene Expression Regulation, Bacterial / Q000235:genetics; D007544:Isopropyl Thiogalactoside / Q000737:chemistry; D010957:Plasmids; D016133:Polymerase Chain Reaction; D011933:Reagent Kits, Diagnostic; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D012097:Repressor Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D016622:Silver Staining; D013056:Spectrophotometry, Ultraviolet; D013752:Tetracycline / Q000037:antagonists & inhibitors* / Q000378:metabolism
D001426:Bacterial Proteins; D002457:Cell Extracts; D017931:DNA Primers; D011933:Reagent Kits, Diagnostic; D011994:Recombinant Proteins; D012097:Repressor Proteins; C034811:tetracycline resistance-encoding transposon repressor protein; D007544:Isopropyl Thiogalactoside; D013752:Tetracycline; D000645:Ammonium Sulfate