title: New method for purifying histidine-rich glycoprotein from human plasma redefines its functional properties.
abstract: Histidine-rich glycoprotein (HRG) is a relatively abundant plasma protein that has been implicated in multiple biological processes including immunity, tumor progression, and vascular biology. However, current protocols for purifying HRG from plasma result in the copurification of contaminating proteins and raise questions over the validity of biological activities ascribed to HRG. In this study, we describe a two-step protocol for the large-scale purification of HRG from human plasma using a combination of metal affinity and ion exchange chromatography. The protocol employs a rapid and simple strategy to isolate highly purified HRG that minimizes proteolytic cleavage of the protein. The purification of HRG was assessed at each stage by measuring the amount of HRG immunoreactive protein using a specific monoclonal antibody against total protein, and demonstrated ~1,000-fold purification with an overall yield of ~32%. Mass spectrometry analysis demonstrated that plasma-derived HRG was free of contaminating proteins and gel electrophoresis showed it to have minimal proteolytic degradation. Characterization of protein by physical method showed that the protein exists as a single, monodisperse species. In contrast to the previous studies of HRG purified by different methods, HRG purified using the new procedure demonstrated a reduced profile of functions. Although the HRG retained binding to heparin and phosphatidic acid, it did not interact with necrotic cells or other cellular lipids. These data demonstrate that HRG does not exhibit the broad interactive properties that have been reported previously, suggesting that copurification of HRG-binding partners or other impurities are responsible for some of the reported functional properties. The findings in this study demonstrate that the new purification procedure can provide a ready source of pure HRG to assess ligand specificity and biological function of this important plasma protein.
authors.affiliation: La Trobe Institute for Molecular Science, La Trobe University, Melbourne, VIC, Australia.
authors.forename: Kruti K
authors.initials: KK
authors.lastname: Patel
authors.forename: Ivan K H
authors.initials: IK
authors.lastname: Poon
authors.forename: Gert H
authors.initials: GH
authors.lastname: Talbo
authors.forename: Matthew A
authors.initials: MA
authors.lastname: Perugini
authors.forename: Nicole L
authors.initials: NL
authors.lastname: Taylor
authors.forename: Troy J
authors.initials: TJ
authors.lastname: Ralph
authors.forename: Nicholas J
authors.initials: NJ
authors.lastname: Hoogenraad
authors.forename: Mark D
authors.initials: MD
authors.lastname: Hulett
mesh_terms: D000595:Amino Acid Sequence; D016477:Artifacts; D002482:Cellulose / Q000031:analogs & derivatives / Q000737:chemistry; D002852:Chromatography, Ion Exchange; D002942:Circular Dichroism; D006801:Humans; D008563:Membrane Lipids / Q000737:chemistry; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry; D011485:Protein Binding; D017433:Protein Structure, Secondary; D011506:Proteins / Q000737:chemistry / Q000302:isolation & purification* / Q000502:physiology*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D000099053:Histidine-Rich Glycoprotein
chemical_list: D008563:Membrane Lipids; D010446:Peptide Fragments; D011506:Proteins; D000099053:Histidine-Rich Glycoprotein; D002482:Cellulose; C008881:phosphocellulose
keywords: histidine-rich glycoprotein (HRG); metal affinity chromatography; phosphatidic acid; plasma protein; protein purification