Expression and purification of epidermal cell differentiation inhibitor (EDIN) from Bacillus subtilis.
The expression of staphylococcal epidermal cell differentiation inhibitor (EDIN), an ADP-ribosyltransferase targeting the small GTP-binding protein rho p21, was examined using Bacillus subtilis. A recombinant plasmid, containing B. licheniformis alpha-amylase promoter flanking either 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 and ED8, respectively. ED7 extracellularly produced recombinant protein, which was purified to homogeneity through column chromatography using SP-Toyopearl 650 cation-exchange gel and the HA1000 hydroxyapatite HPLC column. ED8 did not grow in broth culture. Biochemical and biological studies of purified protein revealed that ED7 produced a correctly processed recombinant EDIN, indistinguishable from natural EDIN.
Hatazaki
K
K
Department of Microbiology, Hiroshima University School of Dentistry, Japan.
Sugai
M
M
Momota
H
H
Ohgai
H
H
Komatsuzawa
H
H
Suginaka
H
H
D016475:3T3 Cells; D000244:Adenosine Diphosphate / Q000378:metabolism*; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D001412:Bacillus subtilis / Q000737:chemistry*; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D001483:Base Sequence; D015153:Blotting, Western; D002522:Chlorocebus aethiops; D002851:Chromatography, High Pressure Liquid; D017931:DNA Primers / Q000737:chemistry; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D007700:Kinetics; D051379:Mice; D010957:Plasmids; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification; D013211:Staphylococcus aureus / Q000737:chemistry; D013997:Time Factors; D014709:Vero Cells
D000596:Amino Acids; D001426:Bacterial Proteins; D017931:DNA Primers; D011994:Recombinant Proteins; C067120:epidermal cell differentiation inhibitor, Staphylococcus aureus; D000244:Adenosine Diphosphate