title: Purification of Lac repressor protein using polymer displacement and immobilization of the protein.
abstract: Lac repressor protein was purified from E. coli BMH8117 harboring plasmid pWB1000 and E. coli K12BMH 71-18 strains. Displacement of the protein with poly(ethyleneimine) (PEI) from phosphocellulose cation exchange column was shown to be an effective elution strategy. It resulted in better recoveries and sharper elution profiles than traditional salt elution without effecting the purity of the protein. The elution is assumed to proceed via displacement of bound protein by PEI when the polymer binds to the ion exchanger. The minor impurities in the protein solution were finally removed by chromatography on immobilized metal affinity column. The repressor protein undergoes distinct conformational changes upon addition of specific inducer isopropyl-beta-D-thiogalactoside (IPTG), which is evidenced by changes in ultraviolet absorption spectrum. The protein was immobilized covalently to the Sepharose matrix. The intact biological activity of the protein after immobilization was shown by binding of genomic DNA and lac operator plasmid DNA from E. coli to the immobilized lac repressor.
authors.affiliation: Department of Biotechnology, Lund University, Sweden.
authors.forename: A
authors.initials: A
authors.lastname: Kumar
authors.lastname: Galaev IYu
authors.forename: B
authors.initials: B
authors.lastname: Mattiasson
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry / Q000302:isolation & purification*; D002853:Chromatography, Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000737:chemistry / Q000235:genetics; D029968:Escherichia coli Proteins; D056968:Lac Repressors; D011108:Polymers; D012097:Repressor Proteins / Q000737:chemistry / Q000302:isolation & purification*
chemical_list: D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D056968:Lac Repressors; C539064:LacI protein, E coli; D011108:Polymers; D012097:Repressor Proteins