title: Identification of native Escherichia coli BL21 (DE3) proteins that bind to immobilized metal affinity chromatography under high imidazole conditions and use of 2D-DIGE to evaluate contamination pools with respect to recombinant protein expression level.
abstract: Immobilized metal affinity chromatography (IMAC) is a widely used purification tool for the production of active, soluble recombinant proteins. Escherichia coli proteins that routinely contaminate IMAC purifications have been characterized to date. The work presented here narrows that focus to the most problematic host proteins, those retaining nickel affinity under elevated imidazole conditions, using a single bind-and-elute step. Two-dimensional difference gel electrophoresis, a favored technique for resolving complex protein mixtures and evaluating their expression, here discerns variation in the soluble extract pools that are loaded in IMAC and the remaining contaminants with respect to varied levels of recombinant protein expression. Peptidyl-prolyl isomerase SlyD and catabolite activator protein (CAP) are here shown to be the most persistent contaminants and have greater prevalence at low target protein expression.
authors.affiliation: Department of Bioengineering, 306 Center for Biotechnology, 300 Technology Drive, University of Pittsburgh, Pittsburgh, PA 15219, USA.
authors.forename: Patrick
authors.initials: P
authors.lastname: Bartlow
authors.forename: Guy T
authors.initials: GT
authors.lastname: Uechi
authors.forename: John J
authors.initials: JJ
authors.lastname: Cardamone
authors.forename: Tamanna
authors.initials: T
authors.lastname: Sultana
authors.forename: McKinzie
authors.initials: M
authors.lastname: Fruchtl
authors.forename: Robert R
authors.initials: RR
authors.lastname: Beitle
authors.forename: Mohammad M
authors.initials: MM
authors.lastname: Ataai
mesh_terms: D001709:Biotechnology / Q000592:standards*; D002846:Chromatography, Affinity / Q000379:methods*; D002373:Cyclic AMP Receptor Protein / Q000032:analysis / Q000737:chemistry; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods*; D029968:Escherichia coli Proteins / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism; D007093:Imidazoles / Q000737:chemistry*; D009532:Nickel / Q000378:metabolism; D019696:Peptidylprolyl Isomerase / Q000032:analysis / Q000737:chemistry; D011994:Recombinant Proteins / Q000592:standards*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
chemical_list: D002373:Cyclic AMP Receptor Protein; D029968:Escherichia coli Proteins; D007093:Imidazoles; D011994:Recombinant Proteins; C085606:SlyD protein, E coli; C029899:imidazole; D009532:Nickel; D019696:Peptidylprolyl Isomerase