title: L-arginine mediated renaturation enhances yield of human, α6 Type IV collagen non-collagenous domain from bacterial inclusion bodies.
abstract: The anti-angiogenic, carboxy terminal non-collagenous domain (NC1) derived from human Collagen type IV alpha 6 chain, [α6(IV)NC1] or hexastatin, was earlier obtained using different recombinant methods of expression in bacterial systems. However, the effect of L-arginine mediated renaturation in enhancing the relative yields of this protein from bacterial inclusion bodies has not been evaluated. In the present study, direct stirring and on-column renaturation methods using L-arginine and different size exclusion chromatography matrices were applied for enhancing the solubility in purifying the recombinant α6(IV)NC1 from bacterial inclusion bodies. This methodology enabled purification of higher quantities of soluble protein from inclusion bodies, which inhibited endothelial cell proliferation, migration and tube formation. Thus, the scope for L-arginine mediated renaturation in obtaining higher yields of soluble, biologically active NC1 domain from bacterial inclusion bodies was evaluated.
authors.affiliation: Cell Signaling, Retinal and Tumor Angiogenesis Laboratory, Department of Genetics, Boys Town National Research Hospital, Omaha, NE 68131, USA.
authors.forename: Venugopal
authors.initials: V
authors.lastname: Gunda
authors.forename: Chandra Shekhar
authors.initials: CS
authors.lastname: Boosani
authors.forename: Raj Kumar
authors.initials: RK
authors.lastname: Verma
authors.forename: Chittibabu
authors.initials: C
authors.lastname: Guda
authors.forename: Yakkanti Akul
authors.initials: YA
authors.lastname: Sudhakar
mesh_terms: D020533:Angiogenesis Inhibitors / Q000302:isolation & purification / Q000378:metabolism*; D001120:Arginine / Q000378:metabolism*; D002465:Cell Movement / Q000502:physiology*; D049109:Cell Proliferation; D002478:Cells, Cultured; D002850:Chromatography, Gel; D003001:Cloning, Molecular; D024141:Collagen Type IV / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D004731:Endotoxins / Q000378:metabolism; D061307:Human Umbilical Vein Endothelial Cells / Q000166:cytology / Q000378:metabolism*; D006801:Humans; D002479:Inclusion Bodies; D020673:Protein Renaturation; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D012995:Solubility
chemical_list: D020533:Angiogenesis Inhibitors; D024141:Collagen Type IV; D004731:Endotoxins; D011994:Recombinant Proteins; D001120:Arginine