Figure 5

Purification of hydrogenase from E. coli over-expressing wild type A. macleodii hydrogenase (pIY085) or H230C/P285C doubly-substituted enzyme (pIY086). Associated enzyme activities can be found in Table S1. Samples are either cleared lysates (left) or Streptactin-purified proteins (right) as indicated at the bottom of the figure. Top: anti-HynL western blot. Middle: anti-Strep western blot, detecting HynS-strep protein. Bottom: Sypro-Ruby stain of a parallel gel. pRC41-4 and pCM012 are constructs expressing wild type and doubly-substituted enzymes, respectively, and are included as negative controls for the strep-tag blot. pIY085 and pIY086, respectively, are the equivalent constructs, except bearing a C-terminal strep-tag on the HynS protein.