title: A novel gigaporous GSH affinity medium for high-speed affinity chromatography of GST-tagged proteins.
abstract: Novel GSH-AP (phenoxyl agarose coated gigaporous polystyrene, Agap-co-PSt) microspheres were successfully prepared by introducing GSH ligand into hydrophilic AP microspheres pre-activated with 1,4-butanediol diglycidyl ether. The gigaporous structure and chromatographic properties of GSH-AP medium were evaluated and compared with commercial GSH Sepharose FF (GSH-FF) medium. The macropores (100-500nm) of gigaporous PSt microspheres were well maintained after coating with agarose and functionalized with GSH ligand. Hydrodynamic experiments showed that GSH-AP column had less backpressure and plate height than those of GSH-FF column at high flow velocity, which was beneficial for its use in high-speed chromatography. The presence of flow-through pores in GSH-AP microspheres also accelerated the mass transfer rate of biomolecules induced by convective flow, leading to high protein resolution and high dynamic binding capacity (DBC) of glutathione S-transferase (GST) at high flow velocity. High purity of GST and GST-tagged recombinant human interleukin-1 receptor antagonist (rhIL-1RA) were obtained from crude extract with an acceptable recovery yield within 1.5min at a velocity up to 1400cm/h. GSH-AP medium is promising for high-speed affinity chromatography for the purification of GST and GST-tagged proteins.
authors.affiliation: National Key Laboratory of Biochemical Engineering, Institute of Process Engineering, Chinese Academy of Sciences, Beijing 100190, PR China.
authors.forename: Yongdong
authors.initials: Y
authors.lastname: Huang
authors.affiliation: National Key Laboratory of Biochemical Engineering, Institute of Process Engineering, Chinese Academy of Sciences, Beijing 100190, PR China.
authors.forename: Rongyue
authors.initials: R
authors.lastname: Zhang
authors.affiliation: National Key Laboratory of Biochemical Engineering, Institute of Process Engineering, Chinese Academy of Sciences, Beijing 100190, PR China.
authors.forename: Juan
authors.initials: J
authors.lastname: Li
authors.affiliation: National Key Laboratory of Biochemical Engineering, Institute of Process Engineering, Chinese Academy of Sciences, Beijing 100190, PR China.
authors.forename: Qiang
authors.initials: Q
authors.lastname: Li
authors.affiliation: National Key Laboratory of Biochemical Engineering, Institute of Process Engineering, Chinese Academy of Sciences, Beijing 100190, PR China.
authors.forename: Zhiguo
authors.initials: Z
authors.lastname: Su
authors.affiliation: National Key Laboratory of Biochemical Engineering, Institute of Process Engineering, Chinese Academy of Sciences, Beijing 100190, PR China. Electronic address: ghma@home.ipe.ac.cn.
authors.forename: Guanghui
authors.initials: G
authors.lastname: Ma
mesh_terms: D000327:Adsorption; D002846:Chromatography, Affinity / Q000295:instrumentation* / Q000379:methods; D005982:Glutathione Transferase / Q000235:genetics / Q000378:metabolism*; D019995:Laboratory Chemicals / Q000737:chemistry; D008863:Microspheres; D016062:Porosity; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism
chemical_list: D019995:Laboratory Chemicals; D011993:Recombinant Fusion Proteins; D005982:Glutathione Transferase
keywords: GSH affinity chromatography; GST-tagged proteins; Gigaporous polystyrene microspheres; High-speed chromatography