title: [Purification of glutathione-S-transferase fusion protein by glutathione coupled magnetic particles].
abstract: We established a purification system for glutathione-S-transferase (GST) fusion protein using glutathione coupled magnetic particle. Glutathione was coupled covalently to the surface of magnetic particles with isothiocyanate functional groups. Cell lysate, containing the fusion protein, was then incubated with these glutathione coupled magnetic particles at room temperature. Unbound and non-specifically bound proteins were removed by wash steps. Subsequently, the GST-fusion protein was eluted from the magnetic particles by the addition of reduced glutathione. The resulting fusion protein was tested for purity using SDS-PAGE and demonstrated by Western blotting. The concentration of the fusion protein was measured by Bradford method. Both the conditions for incubation and washing were optimized. The results showed that 150 microg glutathione could be bound on 1 mg of particle surface and 10 mg of the glutatione-coupled magnetic particles was suitable for 100 microL lysate, the optimal incubation time for reaction between particles and lysate was 40 min. The magnetic particles could help purify efficiently GST-fusion protein with a yield of around 516 microg fusion protein per 10 mg particles. Magnetic particles can be successfully used in a simple, rapid and reliable method for the purification of GST-fusion proteins.
authors: School of Life Sciences, Northwest University, Xi'an 710069, China.
authors: Jingjing
authors: J
authors: Zhu
authors: Liu
authors: L
authors: Yang
authors: Lei
authors: L
authors: Yang
authors: Chao
authors: C
authors: Chen
authors: Yali
authors: Y
authors: Cui
mesh_terms: D005978:Glutathione / Q000737:chemistry*; D005982:Glutathione Transferase / Q000737:chemistry* / Q000302:isolation & purification; D058185:Magnetite Nanoparticles / Q000737:chemistry*; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000302:isolation & purification*
chemical_list: D058185:Magnetite Nanoparticles; D011993:Recombinant Fusion Proteins; D005982:Glutathione Transferase; D005978:Glutathione