Journal: PLoS ONE
Article Title: High-Yield Expression of Heterologous [FeFe] Hydrogenases in Escherichia coli
doi: 10.1371/journal.pone.0015491
Figure Lengend Snippet: All data are for cultures of E. coli strain BL21(DE3) ΔiscR , and both iron and cysteine were included in the growth medium. (Fig. 1A) Optical density at 600 nm (shown on a logarithmic scale) of cultures during aerobic (orange circles) and anaerobic (blue circles) growth phases for cells containing the pACYCDuet-1– hydGX – hydEF and pET-21(b) shydA1*–Strep-tag II plasmids. The pH of culture media (×) was also measured. Data for cultures with cells containing the pET-21(b) shydA–Strep-tag II plasmid instead of pET-21(b) shydA1*–Strep-tag II were similar and are not shown. (Fig. 1B) Cell lysate-based hydrogenase activities (µmol MV reduced·min −1 ·mg −1 total protein) for active CpI (red squares) and HydA1 hydrogenase (green triangles) were determined using the methyl viologen reduction assay. Data are the average for n = 3 cultures examined ± standard deviations. (Fig. 1C) SDS-PAGE analysis for the soluble fractions of final cell lysates after the anoxic co-expression of HydA1 or CpI and the HydE, HydF, and HydG maturases: (Lane 1) the molecular weight markers are from the Mark12 TM protein ladder (Invitrogen); (Lane 2) soluble cell lysate protein content for E. coli strain BL21(DE3) ΔiscR following expression of no heterologous proteins from recombinant DNA plasmids; (Lane 3) co-expression of only the HydE, HydF, and HydG maturases; (Lane 4) co-expression of HydE, HydF, HydG, and HydA1– Strep -tag II; and (Lane 5) co-expression of HydE, HydF, HydG, and CpI– Strep -tag II.
Article Snippet: PCR products were then cloned into the pET-21(b) expression vector (Novagen).
Techniques: Strep-tag, Plasmid Preparation, SDS Page, Expressing, Molecular Weight, Recombinant
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