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Proteins, Expression, Isolation and Analysis> | SDS-PAGE results of the overexpressed target proteins. Lane 1, a blank control of induced E. coli BL21(DE3)(pET28a); lane 2, BL21(DE3)(pET-GroES); lane 3, BL21(DE3)(pET-GroEL2); lane 4, BL21(DE3)(pET-rTHS), lane 5, BL21(DE3)(pET-NHase); lane 6, protein size marker (Genscript PAGE-MASTER Protein Standard). Arrows and horizontal lines at left side indicate target protein locations: rTHS, ∼60 kDa; GroEL2, ∼56 kDa; β and α subunits of NHase, ∼25 kDa and 23 kDa; GroES, ∼10 kDa. The extra bands (∼50 kDa, ∼30 kDa) in lane 4 were speculated to be degradation products of rTHS according to protein mass spectrometryanalysis (see Supplementary Fig. S6 for details). | Get A Quote |
Rhodococcus ruber harboring intracellular nitrile hydratase (NHase) is widely used in large-scale acrylamide production. Transcriptome analyses of R. ruber under urea induction and heat shock revealed the novel chaperones GroEL2 and GroES. M. jannaschii chaperone rTHS, functional in organic solvent as reported in literature, was selected as control. In vitro experiments (70 °C/90 °C incubation) showed that GroEL2 from R. ruber was highly thermostable and can stabilize other proteins as well. GroEL2 was co-expressed with NHase in E. coli in three ways: (a) monocistronic expression with one T7 promoter, (b) bicistronic expression with double T7 promoters, and (c) fusion expression with one T7 promo... More