title: Immobilized metal affinity chromatography co-purifies TGF-β1 with histidine-tagged recombinant extracellular proteins.
abstract: Extracellular recombinant proteins are commonly produced using HEK293 cells as histidine-tagged proteins facilitating purification by immobilized metal affinity chromatography (IMAC). Based on gel analyses, this one-step purification typically produces proteins of high purity. Here, we analyzed the presence of TGF-β1 in such IMAC purifications using recombinant extracellular fibrillin-1 fragments as examples. Analysis of various purified recombinant fibrillin-1 fragments by ELISA consistently revealed the presence of picomolar concentrations of active and latent TGF-β1, but not of BMP-2. These quantities of TGF-β1 were not detectable by Western blotting and mass spectrometry. However, the amounts of TGF-β1 were sufficient to consistently trigger Smad2 phosphorylation in fibroblasts. The purification mechanism was analyzed to determine whether the presence of TGF-β1 in these protein preparations represents a specific or non-specific co-purification of TGF-β1 with fibrillin-1 fragments. Control purifications using conditioned medium from non-transfected 293 cells yielded similar amounts of TGF-β1 after IMAC. IMAC of purified TGF-β1 and the latency associated peptide showed that these proteins bound to the immobilized nickel ions. These data clearly demonstrate that TGF-β1 was co-purified by specific interactions with nickel, and not by specific interactions with fibrillin-1 fragments. Among various chromatographic methods tested for their ability to eliminate TGF-β1 from fibrillin-1 preparations, gel filtration under high salt conditions was highly effective. As various recombinant extracellular proteins purified in this fashion are frequently used for experiments that can be influenced by the presence of TGF-β1, these findings have far-reaching implications for the required chromatographic schemes and quality controls.
authors.affiliation: Department of Anatomy and Cell Biology, McGill University, Montreal, Quebec, Canada.
authors.forename: Jasvir
authors.initials: J
authors.lastname: Kaur
authors.forename: Dieter P
authors.initials: DP
authors.lastname: Reinhardt
mesh_terms: D015153:Blotting, Western; D002478:Cells, Cultured; D002675:Child, Preschool; D002846:Chromatography, Affinity / Q000379:methods; D004797:Enzyme-Linked Immunosorbent Assay; D016326:Extracellular Matrix Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D000071838:Fibrillin-1; D000071837:Fibrillins; D057809:HEK293 Cells; D006639:Histidine / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D055767:Immobilized Proteins / Q000235:genetics / Q000378:metabolism; D008297:Male; D008670:Metals / Q000378:metabolism*; D008840:Microfilament Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D009532:Nickel / Q000378:metabolism; D010446:Peptide Fragments / Q000235:genetics / Q000378:metabolism; D010766:Phosphorylation / Q000187:drug effects; D011485:Protein Binding; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism* / Q000494:pharmacology; D051899:Smad2 Protein / Q000378:metabolism; D051900:Smad3 Protein / Q000378:metabolism; D053773:Transforming Growth Factor beta1 / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology; D054392:Adipokines
chemical_list: D016326:Extracellular Matrix Proteins; D000071838:Fibrillin-1; D000071837:Fibrillins; D006639:Histidine; D055767:Immobilized Proteins; D008670:Metals; D008840:Microfilament Proteins; D009532:Nickel; D010446:Peptide Fragments; D011994:Recombinant Proteins; D051899:Smad2 Protein; D051900:Smad3 Protein; D053773:Transforming Growth Factor beta1; C000605722:FBN1 protein, human; D054392:Adipokines