Extended Data Fig. 1: Purification of recombinant IspH proteins from multiple microbial species and measurement of their biochemical activity by methyl viologen assay.
From: RETRACTED ARTICLE: IspH inhibitors kill Gram-negative bacteria and mobilize immune clearance
a, Coomassie-stained gels showing IPTG induction of recombinant 6His-tagged P. falciparum (Pf), E. coli (Ec), P. aeruginosa (Pa) and M. tuberculosis (MTB) IspH. b, Anti-His-tag immunoblots showing the respective purified IspH proteins. Images in a, b are representative of 3 independent purification attempts. c, IspH uses methyl viologen (MV) as an electron donor for the reductive dehydroxylation of HMBPP. Colourless oxidized methyl viologen is restored to its reduced blue form by sodium dithionite. In the absence or inhibition of IspH activity, methyl viologen stays blue. d, e, Methyl viologen assays measuring IspH activity using different concentrations of E. coli IspH at 10 min in the presence of 1 mM HMBPP (d) and different concentrations of HMBPP (e) at 30 min in the presence of 50 nM E. coli IspH. For d, e, data are mean of 3 independent experiments ± s.e.m. ***P < 0.001, **P < 0.01, *P < 0.05, ns, not significant; two-tailed unpaired Student’s t-test, relative to 0 nM IspH in d or 0 μM HMBPP in e.
