EXPRESSION AND PURIFICATION OF EPIDERMAL-CELL DIFFERENTIATION INHIBITOR (EDIN) FROM BACILLUS-SUBTILIS

The expression of staphylococcal epidermal cell differentiation inhibi tor (EDIN), an ADP-ribosyltransferase targeting the small GTP-binding protein rho p21, was examined using Bacillus subtilis. A recombinant p lasmid, containing B. licheniformis alpha-amylase promoter flanking ei ther a beta-glucanase or a B. cereus sphingomyelinase signal sequence, and a DNA fragment corresponding to mature EDIN were constructed and used to transform B. subtilis KN2. Transformants were designated ED7 a nd ED8, respectively. ED7 extracellularly produced recombinant protein , which was purified to homogeneity through column chromatography usin g SP-Toyopearl 650 cation-exchange gel and the HA1000 hydroxyapatite H PLC column. ED8 did not grow in broth culture. Biochemical and biologi cal studies of purified protein revealed that ED7 produced a correctly processed recombinant EDIN, indistinguishable from natural EDIN. (C) 1998 Academic Press.