Purification of His-tagged proteins using Ni2+-poly(2-acetamidoacrylic acid) hydrogel.
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.
Ha
Eun-Ju
EJ
Department of Polymer Science and Engineering, Pusan National University, Busan, Republic of Korea.
Kim
Yu-Jin
YJ
An
Seong Soo A
SS
Kim
Young-Rok
YR
Lee
Jang-Oo
JO
Lee
Sun-Gu
SG
Paik
Hyun-Jong
HJ
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
D000180:Acrylic Resins; D020100:Hydrogels; C535161:poly(2-acetamidoacrylic acid); D049452:Green Fluorescent Proteins; D006639:Histidine