title: Purification of His-tagged proteins using Ni2+-poly(2-acetamidoacrylic acid) hydrogel.
abstract: In this study, a new matrix for immobilized metal affinity chromatography (IMAC) using poly(2-acetamidoacrylic acid) (PAAA) hydrogels complexed with Ni(2+) was developed for the purification of the recombinant histidine-tagged green fluorescence protein (His6-GFP). The Ni(2+)-complexed PAAA hydrogel was prepared by polymerizing 2-acetamidoacrylic acid (AAA) and 2,2'-[(1,4-dioxo-1,4-butanediyl)diamino] bis(2-propenoic acid) (DBDBPA) with potassium persulfate in DMSO, followed by Ni(2+) complexation. Confocal laser scanning microscopy was used to determine the binding of His6-GFP to the Ni(2+)-PAAA hydrogel in three-dimensional space. Photoluminescence spectroscopy revealed an 81% binding efficiency of His6-GFP to the Ni(2+)-PAAA hydrogel yielded with a recovery of 59%. The specificity of His6-GFP binding to Ni(2+)-PAAA hydrogel was compared with that of the PAAA hydrogel without Ni(2+). His6-GFP was purified directly from the cell lysate with Ni(2+)-PAAA hydrogel matrix but the PAAA hydrogel without Ni(2+) had no effect. The major advantage of the Ni(2+)-PAAA hydrogel system over current methods, such as Ni-nitrilotriacetic acid (NTA) agarose beads, was the simple and low-cost procedure for preparing the matrix.
authors.affiliation: Department of Polymer Science and Engineering, Pusan National University, Busan, Republic of Korea.
authors.forename: Eun-Ju
authors.initials: EJ
authors.lastname: Ha
authors.forename: Yu-Jin
authors.initials: YJ
authors.lastname: Kim
authors.forename: Seong Soo A
authors.initials: SS
authors.lastname: An
authors.forename: Young-Rok
authors.initials: YR
authors.lastname: Kim
authors.forename: Jang-Oo
authors.initials: JO
authors.lastname: Lee
authors.forename: Sun-Gu
authors.initials: SG
authors.lastname: Lee
authors.forename: Hyun-Jong
authors.initials: HJ
authors.lastname: Paik
mesh_terms: D000180:Acrylic Resins / Q000138:chemical synthesis / Q000737:chemistry*; D002846:Chromatography, Affinity / Q000379:methods*; D049452:Green Fluorescent Proteins / Q000737:chemistry*; D006639:Histidine / Q000737:chemistry*; D020100:Hydrogels / Q000737:chemistry*; D018613:Microscopy, Confocal
chemical_list: D000180:Acrylic Resins; D020100:Hydrogels; C535161:poly(2-acetamidoacrylic acid); D049452:Green Fluorescent Proteins; D006639:Histidine