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. 2016 Aug 9;6:30839. doi: 10.1038/srep30839

Figure 2. Production and catalytic activity of G6PDH and HXK2 forizymes.

Figure 2

The forizymes were composed of G6PDH-cIL-MtSEO-F1 and MtSEO-F1 subunits (G6PDH forizymes) or HXK2-HAL-MtSEO-F1 and MtSEO-F1 subunits (HXK2 forizymes) and were produced in S. cerevisiae cells. (a) SDS-PAGE analysis for the verification of G6PDH forizyme production. Control forisomes composed solely of MtSEO-F1 were produced and purified simultaneously. Symbols: *untagged MtSEO-F1, •G6PDH-cIL-MtSEO-F1 fusion. (b) Western blot for the detection of the G6PDH-cIL-MtSEO-F1 fusion protein (•) in purified G6PDH forizymes using a Myc epitope-specific antibody (the Myc epitope is part of the synthetic linker cIL). The antibody did not detect the purified control forisomes composed solely of MtSEO-F1. (c) Activity assay for the purified G6PDH forizymes and MtSEO-F1 control forisomes based on measuring the formation of NADPH by monitoring the absorbance at 340 nm in a G6PDH enzyme assay. (d) SDS-PAGE analysis for the verification of HXK2 forizyme production. Control forisomes composed solely of MtSEO-F1 were produced and purified simultaneously. Symbols: *untagged MtSEO-F1, ▼HXK2-HAL-MtSEO-F1 fusion. (e) Western blot for the detection of the HXK2-HIL-MtSEO-F1 fusion protein (▼) in purified HXK2 forizymes using an HA-specific antibody. The antibody did not detect the purified control forisomes composed solely of MtSEO-F1. (f) Activity assay for the purified HXK2 forizymes and MtSEO-F1 control forisomes based on a coupled reaction with soluble G6PDH leading to the formation of NADPH. Each value was corrected for the blank measurement. Graphs show means ± SD of technical triplicates representing one of three independent forizyme purifications.