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. Author manuscript; available in PMC: 2017 Dec 19.
Published in final edited form as: Nat Chem Biol. 2017 Jun 19;13(9):1022–1028. doi: 10.1038/nchembio.2409

Figure 3. Solubilized DsbB variants promote folding of alkaline phosphatase.

Figure 3

(a) Schematic showing disulfide bond connectivity for E. coli alkaline phosphatase, PhoA (2 disulfide bonds depicted by yellow circles connected by yellow lines). Alkaline phosphatase activity measured in periplasmic (white bars) and cytoplasmic (gray bars) fractions derived from the following strains: wt DHB4(DE3) cells (lacking the native phoA gene) carrying no plasmid (empty) or pBAD18-pPhoA (pPhoA); DHB4(DE3) ΔdsbAB cells carrying no plasmid (empty), pBAD18-pPhoA (pPhoA), pFH273 (cPhoA + cDsbA), or pFH273mut (cPhoA + cDsbA(APHA)), along with a pET21d derivative containing SxDsbB, SxDsbBΔCGZ, SxDsbBΔCGZ(C44A), or H0DsbB1/9b as indicated; and SHuffle cells carrying no plasmid (empty) or pFH-cPhoA (cPhoA). Data is the mean of biological triplicates and the error bars represent the standard error of the mean (SEM). (b) Western blot analysis of the same periplasmic (p) and cytoplasmic (c) fractions assayed in (a) as indicated. Blots were probed with anti-PhoA antibody to detect PhoA (top panel) and anti-GroEL antibody to detect GroEL (bottom panel), which served as a cytoplasmic fractionation marker and loading control. Molecular weight (MW) markers are shown on the left. See Supplementary Figure 10 for uncropped versions of the blot images.